Use of 1H-indol-3-yl-2-oxoacetamide compounds

ABSTRACT

A method of treating cancer including administering to a subject in need thereof an effective amount of a compound of the formula 1: 
                         
wherein R 1 , R 2 , R 3 , R 4 , and n are as defined herein.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/310,711, filed on Dec. 5, 2002 now U.S. Pat. No. 6,903,104, whichclaims priority to U.S. Provisional Application No. 60/337,962, filed onDec. 6, 2001. The contents of both applications are hereby incorporatedby reference in their entireties.

TECHNICAL FIELD

This invention relates to novel heteroatom containing compounds andcompositions thereof, and their use for the prevention and treatment ofdisease.

BACKGROUND

Cancer is the leading or second most common cause of death in advancedcountries. Despite continuing advances in both diagnosis and treatmentregimens, most existing treatment methods have undesirable side effectsand limited efficacy in treating and curing the cancerous diseases ofsolid tumors. Treatment of cancer is complicated by the variety andnumber of mechanisms involved in the formation and metastasis of tumors,many of which are still not well understood. Chemotherapy is, however,still one of the major options available for the first-line treatment incancers such as leukemias and second-line treatment for refractory solidtumors. The mechanism of action of some effective anticancer agentsremains unclear. Most of the currently used anticancer agents are smallmolecule chemicals and need to be administered into patients via aparenteral infusion or bolus injection. Clinical complications with theparenteral administrations have been documented and thus extra cares andcost for hospitalization are essential. Recent efforts in the discoveryof anticancer drugs have been focused on finding orally activeanticancer agents.

Angiogenesis refers to the generation of new blood vessels into a tissueor organ. Under normal physiological conditions, humans or animals onlyundergo angiogenesis in very specific restricted situations. Forexample, angiogenesis is normally observed in wound healing, fetal andembryonal development and formation of the corpus luteum, endometriumand placenta. The control of angiogenesis is a highly regulated systemof angiogenic stimulators and inhibitors. The control of angiogenesishas been found to be altered in certain disease states and, in manycases, the pathological damage associated with the disease is related touncontrolled angiogenesis. Thus, methods and compositions are neededthat are capable of inhibiting angiogenesis and which are easilyadministered. A simple and efficacious method of treatment would bethrough the oral route, which could be distributed in a form that thepatient could self-administer.

The invention presents heterocyclic compounds that exhibit cytotoxic andanticancer activity and that inhibit angiogenesis.

SUMMARY

The invention relates to heteroatom containing compounds, compositionsincluding the compounds, and the methods of using the compounds andcompound compositions. The compounds and compositions are useful fortreating diseases or disease symptoms (e.g., cancer, inhibition ofangiogenesis). The invention also provides for methods of making thecompounds.

The invention is based on the discovery that certain heteroatomcontaining compounds have potent anticancer and cytotoxic activity andinhibit angiogenesis. Thus, this invention relates to novel heterocycliccompounds and to their uses in the medical treatment of disease. Theinvention is further based on the discovery that certain indol-3-yloxoacetamido compounds have potent anticancer and cytotoxic activity andare useful in the treatment of a variety of cancers. The invention isfurther based on the discovery that certain indol-3-yl oxoacetamidocompounds have potent anti-angiogenic activity and are useful in thetreatment of a variety of angiogenesis-mediated diseases.

In one aspect, the invention relates to a compound of the followingformula:

wherein,

-   -   each R¹ is independently isoxazolyl, thiazolyl, isothiazolyl,        1,3,4-thiadiazolyl, 1,3-benzothiazolyl, quinolyl, isoquinolyl,        thionaphthenyl, or benzofuranyl, each being optionally        substituted with 1-6 independent R⁵; or when taken together with        R² and the nitrogen atom to which they are attached form a 5-8        membered ring comprising C, N, S, or O atoms wherein any atom is        optionally substituted with an independent R⁵;    -   each R² is independently H, C1-C10 alkyl, or aryl, each being        optionally substituted with 1-4 independent R⁵; or when taken        together with R¹ and the nitrogen atom to which they are        attached form a 5-8 membered ring comprising C, N, S, or O atoms        wherein any atom is optionally substituted with an independent        R⁵;    -   each R³ is independently C1-C10 alkyl, C2-C10 alkenyl, C2-C10        alkynyl, C3-C10 cycloalkyl, C4-C10 cycloalkenyl, aryl,        heteroaryl, or heterocyclyl, each being optionally substituted        with 1-4 independent R⁵;    -   each R⁴ is independently H, NO₂, halo, CN, R⁷, OR⁷, CO₂R⁷, SR⁷,        NR⁷R⁷, C(O)R⁷, C(O)NR⁷R⁷, OC(O)R⁷, S(O)₂R⁷, S(O)₂NR⁷R⁷;        NR⁷C(O)NR⁷R⁷, NR⁷C(O)R⁷, NR⁷(COOR⁷), NR⁷S(O)₂NR⁷R⁷, or        NR⁷S(O)₂R⁷, S(O)₂OR⁷;    -   each n is 0, 1, 2, 3, or 4;    -   each R⁵ is independently H, C1-C10 alkyl optionally substituted        with 14 independent R⁶, C2-C10 alkenyl, C2-C10 alkynyl, C3-C10        cycloalkyl, aryl optionally substituted with 1-4 independent R⁶,        heteroaryl optionally substituted with 1-4 independent R⁶,        heterocyclyl optionally substituted with 1-4 independent R⁶,        halo, haloalkyl, SR⁷, OR⁷, NR⁷R⁷, COOR⁷, NO₂, CN, C(O)R⁷,        C(O)NR⁷R⁷, OC(O)R⁷, S(O)₂R⁷, S(O)₂OR⁷, S(O)₂NR⁷R⁷, NR⁷C(O)NR⁷R⁷,        NR⁷C(O)R⁷, NR⁷(COOR⁷), NR⁷S(O)₂NR⁷R⁷, or NR⁷S(O)₂R⁷;    -   each R⁶ is independently C1-C10 alkyl, C2-C10 alkenyl, C2-C10        alkynyl, halo, haloalkyl, CN, NO₂, OR⁷, or SO₂R⁷;    -   each R⁷ is independently H, OR⁹, C1-C10 alkyl, C2-C10 alkenyl,        C2-C10 alkynyl, C3-C10 cycloalkyl, aryl, heteroaryl, or        heterocyclyl, each optionally substituted with 1-4 independent        R⁸;    -   each R⁸ is independently H, OH, C1-C10 alkyl, halo, aryl, NO₂,        or CN; and    -   each R⁹ is independently H, C1-C10 alkyl, or aryl; each being        optionally substituted with 1-4 independent OH, halo, CN, NO₂,        or CO₂H

In other aspects the compounds, compositions, or methods are those ofany of the formulae herein, wherein R¹ taken together with R² and thenitrogen atom to which they are attached is not 4-phenyl-piperazin-1-yl,4-(pyridin-4-yl)-piperazin-1-yl, 4-(pyridin-2-yl)-piperazin-1-yl,4-(2-nitro-phenyl)-piperazin-1-yl,4-(3,5-dimethoxy-phenyl)-piperazin-1-yl, or4-[bis-(4-fluoro-phenyl)-methyl]-piperazin-1-yl. This proviso may onlyapply to methods of treating cancer or to the compounds themselves.

In other aspects, the invention is compound of formula 1, describedabove, wherein R¹ is isothiazolyl, isoxazolyl, or thiazolyl, each beingoptionally substituted with 1-4 C1-C10 alkyl; or wherein R¹ is3-methyl-5-isothiazolyl; or wherein each R³ is cyanophenyl,chlorophenyl, 3-methyl-5-isoxazolyl, 3-phenyl-5-isoxazoyl, pyridyl, orthiophenyl; or wherein R¹ is 3-methyl-5-isothiazolyl,3-methyl-5-isoxazolyl, 3-phenyl-5-isoxazolyl, 3-tert-butyl-5-isoxazolyl,4-methyl-1,3-thiazol-2-yl, or 1,4-benzodioxan-6-yl, thiazolyl, and R³ iscyanophenyl, 3-methyl-5-isoxazolyl, or thiophenyl; or wherein R¹ is3-methyl-5-isothiazolyl, 3-methyl-5-isoxazolyl, or3-phenyl-5-isoxazolyl, and R³ is cyanophenyl; or pharmaceuticallyacceptable salt thereof.

In another aspect, the invention is a compound of the following formula:

wherein,

-   -   each R¹ is independently C1-C10 alkyl, C2-C10 alkenyl, C2-C10        alkynyl, C3-C10 cycloakyl, C4-C10 cycloalkenyl, aryl,        heteroaryl, or heterocyclyl, each being optionally substituted        with 1-6 independent R⁵; or when taken together with R² and the        nitrogen atom to which they are attached form a 5-8 membered        ring comprising C, N, S, or O atoms wherein any atom is        optionally substituted with an independent R⁵;    -   each R² is independently H, C1-C10 alkyl, or aryl, each being        optionally substituted with 1-4 independent R⁵; or when taken        together with R¹ and the nitrogen atom to which they are        attached form a 5-8 membered ring comprising C, N, S, or O atoms        wherein any atom is optionally substituted with an independent        R⁵;    -   each R³ is independently cyanophenyl, furanyl, thiophenyl,        isoxazolyl, thiazolyl, imidazolyl, 4,5-dihydro-5-isoxazolyl, or        naphthyl; each being optionally substituted with 1-4 independent        R⁵;    -   each R⁴ is independently H, NO₂, halo, CN, R⁷, OR⁷, CO₂R⁷, SR⁷,        NR⁷R⁷, C(O)R⁷, C(O)NR⁷R⁷, OC(O)R⁷, S(O)₂R⁷, S(O)₂NR⁷R⁷,        NR⁷C(O)NR⁷R⁷, NR⁷C(O)R⁷, NR⁷(COOR⁷), NR⁷S(O)₂NR⁷R⁷, or        NR⁷S(O)₂R⁷, S(O)₂OR⁷;    -   each n is 0, 1, 2, 3, or 4;    -   each R⁵ is independently H, C1-C10 alkyl optionally substituted        with 1-4 independent R⁶, C2-C10 alkenyl, C2-C10 alkynyl, C3-C10        cycloalkyl, aryl optionally substituted with 1-4 independent R⁶,        heteroaryl optionally substituted with 1-4 independent R⁶,        heterocyclyl optionally substituted with 1-4 independent R⁶,        halo, haloalkyl, SR⁷, OR⁷, NR⁷R⁷, COOR⁷, NO₂, CN, C(O)R⁷,        C(O)NR⁷R⁷, OC(O)R⁷, S(O)₂R⁷, S(O)₂OR⁷, S(O)₂NR⁷R⁷, NR⁷C(O)NR⁷R⁷,        NR⁷C(O)R⁷, NR⁷(COOR⁷), NR⁷S(O)₂NR⁷R⁷, or NR⁷S(O)₂R⁷;    -   each R⁶ is independently C1-C10 alkyl, C2-C10 alkenyl, C2-C10        alkynyl, C3-C10 cycloalkyl, halo, haloalkyl, CN, NO₂, OR⁷, or        SO₂R⁷;    -   each R⁷ is independently H, C1-C10 alkyl, C2-C10 alkenyl, C2-C10        alkynyl; C3-C10 cycloalkyl; aryl, heteroaryl, or heterocyclyl,        each optionally substituted with 1-4 independent R⁸;    -   each R⁸ is independently H, OH, OR⁹, C1-C10 alkyl, halo, aryl,        NO₂, or CN; and    -   each R⁹ is independently H, C1-C10 alkyl, or aryl; each being        optionally substituted with 1-4 independent OH, halo, CN, NO₂,        or CO₂H;

wherein R¹ taken together with R² and the nitrogen atom to which theyare attached is not 4-phenyl-piperazin-1-yl,4-(pyridin-4-yl)-piperazin-1-yl, 4-(pyridin-2-yl)-piperazin-1-yl,4-(2-nitro-phenyl)-piperazin-1-yl,4-(3,5-dimethoxy-phenyl)-piperazin-1-yl, or4-[bis-(4-fluoro-phenyl)-methyl]-piperazin-1-yl.

In other aspects, the invention is a compound of formula 1, wherein eachR³ is cyanophenyl, furanyl, or thiophenyl; or wherein each R3 isthiophenyl; or wherein each R¹ is 3-methyl-5-isothiazolyl,1,4-benzodioxan-6-yl or 6-quinolyl; or wherein each R¹ is3-methyl-5-isothiazolyl, pyridyl, halophenyl, or methoxyphenyl, and R³is 3-alkyl-5-isoxazolyl, 3-aryl-5-isoxazolyl, or cyanophenyl; or whereineach R¹ is 3-methyl-5-isothiazolyl or 6-quinolyl, and R³ is furanyl,thiophenyl, or 1-methyl-1H-5-imidazolyl.

In another aspect, the invention is a formulation including a compoundof any of the formulae herein, and an excipient suitable foradministration to a subject. In another aspect, the invention is acomposition including a compound of any of the formulae herein, and apharmaceutically acceptable carrier.

Additionally, the invention relates to a composition including acompound of any of the formulae herein, a pharmaceutically acceptablecarrier, and an additional therapeutic agent.

The additional therapeutic agent can be an anticancer agent. Theadditional therapeutic agent can also be paclitaxel, docitaxel,doxorubicin, daunorubicin, epirubicin, fluorouracil, melphalan,cis-platin, carboplatin, cyclophosphamide, mitomycin, methotrexate,mitoxantrone, vinblastine, vincristine, ifosfamide, teniposide,etoposide, bleomycin, leucovorin, cytarabine, dactinomycin, interferonalpha, streptozocin, prednisolone, or procarbazine.

In another aspect, the invention is a method of treating a subject(e.g., human, mammal, dog, cat, horse) having cancer includingadministering to the subject an effective amount of a compound of any ofthe formulae herein.

The cancer can be a human leukemia, sarcoma, osteosarcoma, lymphoma,melanoma, ovarian, skin, testicular, gastric, pancreatic, renal, breast,prostate colorectal, head and neck, brain, esophageal, bladder, adrenalcortical, lung, bronchus, endometrial, cervical or hepatic cancer, orcancer of unknown primary site. The cancer can also be a cancer of adrug resistance phenotype of which the cancer cells expressP-glycoprotein (MDR), multidrug resistance-associated proteins (MRP),lung cancer resistance-associated proteins (LRP), breast cancerresistance proteins (BCRP) or other proteins associated with resistanceto anticancer drugs.

In another aspect, the invention is a method of inhibiting angiogenesisin a subject identified as in need thereof, including administering tothe subject an effective amount of a compound of any of the formulaeherein.

In still another aspect, the invention is a method of treating diseaseor disease symptoms in a subject, including administration to thesubject an effective amount of a compound of any formulae herein. Thedisease can be angiogenesis-mediated, associated with cornealneovascularization, inflammatory, or any of those specifically recitedherein.

Angiogenesis-mediated diseases include choroidal neovascular diseases,retina neovascular diseases, neovascularization of the angle,Bartonellosis, chronic inflammation, osteoarthritis, atherosclerosisphemphigoid, trachoma, or Osler-Webber-Rendu disease.

Diseases associated with corneal-neovascularization that can be treatedaccording to the present invention include but are not limited to,diabetic retinopathy, retinopathy of prematurity, corneal graftrejection, neovascular glaucoma and retrolental fibroplasia, epidemickeratoconjunctivitis, Vitamin A deficiency, contact lens overwear,atopic keratitis, superior limbic keratitis, pterygium keratitis sicca,sjogrens, acne rosacea, phylectenulosis, syphilis, Mycobacteriainfections, lipid degeneration, chemical burns, bacterial ulcers, fungalulcers, Herpes simplex infections, Herpes zoster infections, protozoaninfections, Kaposi sarcoma, Mooren ulcer, Terrien's marginaldegeneration, mariginal keratolysis, trauma, rheumatoid arthritis,systemic lupus, polyarteritis, Wegeners sarcoidosis, Scieritis, Steven'sJohnson disease, periphigoid radial keratotomy, and corneal graphrejection. Diseases associated with retinal/choroidal neovascularizationthat can be treated according to the present invention include, but arenot limited to, diabetic retinopathy, macular degeneration, sickle cellanemia, sarcoid, syphilis, pseudoxanthoma elasticum, Pagets disease,vein occlusion, artery occlusion, carotid obstructive disease, chronicuveitis/vitritis, mycobacterial infections, Lyme's disease, systemiclupus erythematosis, retinopathy of prematurity, Eales disease, Bechetsdisease, infections causing a retinitis or choroiditis, presumed ocularhistoplasmosis, Bests disease, myopia, optic pits, Stargarts disease,pars planitis, chronic retinal detachment, hyperviscosity syndromes,toxoplasmosis, trauma and post-laser complications. Other diseasesinclude, but are not limited to, diseases associated with rubeosis(neovasculariation of the angle) and diseases caused by the abnormalproliferation of fibrovascular or fibrous tissue including all forms ofproliferative vitreoretinopathy, whether or not associated withdiabetes.

Another disease which can be treated according to the present inventionis rheumatoid arthritis. While not being held to any one particulartheory, it is believed that the blood vessels in the synovial lining ofthe joints undergo angiogenesis. In addition to forming new vascularnetworks, the endothelial cells release factors and reactive oxygenspecies that lead to pannus growth and cartilage destruction. Thefactors involved in angiogenesis may actively contribute to, and helpmaintain, the chronically inflamed state of rheumatoid arthritis.

Such disease states as ulcerative colitis and Crohn's disease showhistological changes with the ingrowth of new blood vessels into theinflamed tissues. Bartonellosis, a bacterial infection found in SouthAmerica, can result in a chronic stage that is characterized byproliferation of vascular endothelial cells. Another pathological roleassociated with angiogenesis is found in atherosclerosis. The plaquesformed within the lumen of blood vessels have been shown to haveangiogenic stimulatory activity

Other diseases that can be treated according to the present inventionare hemangiomas, Osler-Weber-Rendu disease, or hereditary hemorrhagictelangiectasia, solid or blood borne tumors and acquired immunedeficiency syndrome.

The methods described herein can further include identifying the subjectas in need of a particular treatment. Identifying a subject in need ofsuch treatment can be in the judgement of a subject or a health careprofessional and can be subjective (e.g., opinion) or objective (e.g.,measurable by a test or diagnostic method).

In one aspect, the invention is a compound of any of the formulaeherein, wherein each R³ is independently C1-C10 alkyl, C2-C10 alkenyl,C2-C10 alkynyl, C3-C10 cycloalkyl, C4-C10 cycloalkenyl, heteroaryl, orheterocyclyl, each being optionally substituted with 1-4 independent R⁵.

In another aspect, the invention is a compound of any of the formulaherein, wherein each R³ is independently aryl optionally substitutedwith 1-4 independent R⁵.

Another aspect of the invention is a compound of any of the formulaeherein, wherein each R³ is independently C1-C10 alkyl, C2-C10 alkenyl,C2-C10 alkynyl, C3-C10 cycloalkyl, C4-C10 cycloalkenyl, aryl,heteroaryl, or heterocyclyl, each being optionally substituted with 14independent R⁵; wherein R³ is not fluorophenyl.

This invention relates to methods of making compounds of any formulaeherein comprising reacting any one or more of the compounds or formulaedelineated herein including any processes delineated herein.

The invention further relates to a method of making a compound of any ofthe formulae herein, comprising taking an indole derivative of theformula:

wherein each R⁴ and n is independently defined herein, including informula 1;

and reacting it with one or more chemical reagents in one or more stepsto produce a compound of any of the formulae herein.

The invention also related to a method of making a compound of any ofthe formulae herein, including taking an indole derivative of theformula:

wherein

-   -   each R⁴, and n is independently defined herein, including in        formula 1; and    -   each R³ is independently C1-C10 alkyl, C2-C10 alkenyl, C2-C10        alkynyl, C3-C10 cycloalkyl, C4-C10 cycloalkenyl, aryl,        heteroaryl, or heterocyclyl, each being optionally substituted        with 1-4 independent R⁵; wherein each R⁵ is independently        defined herein, including in formula 1; and    -   reacting it with one or more chemical reagents in one or more        steps to produce a compound of any of the formulae herein.

The invention further relates to a method of making a compound of any ofthe formulae herein, including taking an indole derivative of theformula:

wherein

-   -   each R⁴, and n is independently defined herein, including in        formula 1;    -   each R¹ is independently C1-C10 alkyl, C2-C10 alkenyl, C2-C10        alkynyl, C3-C10 cycloakyl, C4-C10 cycloalkenyl, aryl,        heteroaryl, or heterocyclyl, each being optionally substituted        with 1-4 independent R⁵; wherein each R⁵ is independently        defined as herein, including formula 1, or when taken together        with R² and the nitrogen atom to which they are attached form a        5-8 membered ring comprising C, N, S, or O atoms wherein any        atom is optionally substituted with an independent R⁵;    -   each R² is independently H, C1-C10 alkyl, or aryl, each being        optionally substituted with 1-4 independent R⁵ or when taken        together with R¹ and the nitrogen atom to which they are        attached form a 5-8 membered ring comprising C, N, S, or O atoms        wherein any atom is optionally substituted with an independent        R⁵;

wherein R¹ taken together with R² and the nitrogen atom to which theyare attached is not 4-phenyl-piperazin-1-yl,4-(pyridin-4-yl)-piperazin-1-yl, 4-(pyridin-2-yl)-piperazin-1-yl,4-(2-nitro-phenyl)-piperazin-1-yl,4-(3,5-dimethoxy-phenyl)-piperazin-1-yl, or4-[bis-(4-fluoro-phenyl)-methyl]-piperazin-1-yl; and

reacting it with one or more chemical reagents in one or more steps toproduce a compound of any of the formulae herein.

The invention also includes a method of making a compound of Formula 1:

wherein,

-   -   each R¹ is independently isoxazolyl, thiazolyl, isothiazolyl,        1,3,4-thiadiazolyl, 1,3-benzothiazolyl, quinolyl, isoquinolyl,        thionaphthenyl, or benzofuanyl, each being optionally        substituted with 1-6 independent R⁵; or when taken together with        R² and the nitrogen atom to which they are attached form a 5-8        membered ring comprising C, N, S, or O atoms wherein any atom is        optionally substituted with an independent R⁵;    -   each R² is independently H, C1-C10 alkyl, or aryl, each being        optionally substituted with 1-4 independent R⁵; or when taken        together with R¹ and the nitrogen atom to which they are        attached form a 5-8 membered ring comprising C, N, S, or O atoms        wherein any atom is optionally substituted with an independent        R⁵;    -   each R³ is independently C1-C10 alkyl, C2-C10 alkenyl, C2-C10        alkynyl, C3-C10 cycloalkyl, C4-C10 cycloalkenyl, aryl,        heteroaryl, or heterocyclyl, each being optionally substituted        with 1-4 independent R⁵;    -   each R⁴ is independently H, NO₂, halo, CN, R⁷, OR⁷, CO₂R⁷, SR⁷,        NR⁷R⁷, C(O)R⁷, C(O)NR⁷R⁷, OC(O)R⁷, S(O)₂R⁷, S(O)₂NR⁷R⁷,        NR⁷C(O)NR⁷R⁷, NR⁷C(O)R⁷, NR⁷(COOR⁷), NR⁷S(O)₂NR⁷R⁷, or        NR⁷S(O)₂R⁷, S(O)₂OR⁷;    -   each n is 0, 1, 2, 3, or 4;    -   each R⁵ is independently H, C1-C10 alkyl optionally substituted        with 14 independent R⁶, C2-C10 alkenyl, C2-C10 alkynyl, C3-C10        cycloalkyl, aryl optionally substituted with 1-4 independent R⁶,        heteroaryl optionally substituted with 1-4 independent R⁶,        heterocyclyl optionally substituted with 1-4 independent R⁶,        halo, haloalkyl, SR⁷, OR⁷, NR⁷R⁷, COOR⁷, NO₂, CN, C(O)R⁷,        C(O)NR⁷R⁷, OC(O)R⁷, S(O)₂R⁷, S(O)₂OR⁷, S(O)₂NR⁷R⁷, NR⁷C(O)NR⁷R⁷,        NR⁷C(O)R⁷, NR⁷(COOR⁷), NR⁷S(O)₂NR⁷R⁷, or NR⁷S(O)₂R⁷;    -   each R⁶ is independently C1-C10 alkyl, C2-C10 alkenyl, C2-C10        alkynyl, halo, haloalkyl, CN, NO₂, OR⁷, or SO₂R⁷;    -   each R⁷ is independently H, C1-C10 alkyl, C2-C10 alkenyl, C2-C10        alkynyl; C3-C10 cycloalkyl; aryl, heteroaryl, or heterocyclyl,        each optionally substituted with 1-4 independent R⁸;    -   each R⁸ is independently H, OH, OR⁹, C1-C10 alkyl, halo, aryl,        NO₂, or CN; and    -   each R⁹ is independently H, C1-C10 alkyl, or aryl; each being        optionally substituted with 1-4 independent OH, halo, CN, NO₂,        or CO₂H;

wherein R¹ taken together with R² and the nitrogen atom to which theyare attached is not 4-phenyl-piperazin-1-yl,4-(pyridin-4-yl)-piperazin-1-yl, 4-(pyridin-2-yl)-piperazin-1-yl,4-(2-nitro-phenyl)-piperazin-1-yl,4-(3,5-dimethoxy-phenyl)-piperazin-1-yl, or4-[bis-(4-fluoro-phenyl)-methyl]-piperazin-1-yl;

-   -   and reacting with an indole of Formula 2:

-   -   wherein R⁴ and n are each independently as defined above;    -   with a reagent of Formula 3:

-   -   wherein X is independently a leaving group;    -   followed by addition of HNR¹R², where R¹ and R² are        independently as defined above; and reacting the resulting        compound of Formula 4:

-   -   wherein all variables are as defined above;    -   with a base and a compound of the formula XCH₂R³; wherein X is        independently a leaving group and R³ is as defined above;    -   resulting in a compound of Formula 1.

In certain embodiments, X is independently halo, O-triflate, alkoxyl.

The invention also relates a method of making a compound of Formula 1:

wherein,

-   -   each R¹ is independently C1-C10 alkyl, C2-C10 alkenyl, C2-C10        alkynyl, C3-C10 cycloakyl, C4-C10 cycloalkenyl, aryl,        heteroaryl, or heterocyclyl, each being optionally substituted        with 1-4 independent R⁵; or when taken together with R² and the        nitrogen atom to which they are attached form a 5-8 membered        ring comprising C, N, S, or O atoms wherein any atom is        optionally substituted with an independent R⁵;    -   each R² is independently H, C1-C10 alkyl, or aryl, each being        optionally substituted with 1-4 independent R⁵; or when taken        together with R¹ and the nitrogen atom to which they are        attached form a 5-8 membered ring comprising C, N, S, or O atoms        wherein any atom is optionally substituted with an independent        R⁵;    -   each R³ is independently cyanophenyl, furanyl, thiophenyl,        isoxazolyl, thiazolyl, imidazolyl, 4,5-dihydro-5-isoxazolyl, or        naphthyl; each being optionally substituted with 1-4 independent        R⁵;    -   each R⁴ is independently H, NO₂, halo, CN, R⁷, OR⁷, CO₂R⁷, SR⁷,        NR⁷R⁷, C(O)R⁷, C(O)NR⁷R⁷, OC(O)R⁷, S(O)₂R⁷, S(O)₂NR⁷R⁷,        NR⁷C(O)NR⁷R⁷, NR⁷C(O)R⁷, NR⁷(COOR⁷), NR⁷S(O)₂NR⁷R⁷, or        NR⁷S(O)₂R⁷, S(O)₂OR⁷;    -   each n is 0, 1, 2, 3, or 4;    -   each R⁵ is independently H, C1-C10 alkyl optionally substituted        with 14 independent R⁶, C2-C10 alkenyl, C2-C10 alkynyl; C3-C10        cycloalkyl; aryl optionally substituted with 1-4 independent R⁶,        heteroaryl optionally substituted with 1-4 independent R⁶,        heterocyclyl optionally substituted with 1-4 independent R⁶,        halo, haloalkyl, SR⁷, OR⁷, NR⁷R⁷, COOR⁷, NO₂, CN, C(O)R⁷,        C(O)NR⁷R⁷, OC(O)R⁷, S(O)₂R⁷, S(O)₂OR⁷, S(O)₂NR⁷R⁷, NR⁷C(O)NR⁷R⁷,        NR⁷C(O)R⁷, NR⁷(COOR⁷), NR⁷S(O)₂NR⁷R⁷, or NR⁷S(O)₂R⁷;    -   each R⁶ is independently C1-C10 alkyl, C2-C10 alkenyl, C2-C10        alkynyl, halo, haloalkyl, CN, NO₂, OR⁷, or SO₂R⁷;    -   each R⁷ is independently H, C1-C10 alkyl, C2-C10 alkenyl, C2-C10        alkynyl; C3-C10 cycloalkyl; aryl, heteroaryl, or heterocyclyl,        each optionally substituted with 1-4 independent R⁸;    -   each R⁸ is independently H, OH, OR⁹, C1-C10 alkyl, halo, aryl,        NO₂; and    -   each R⁹ is independently H, C1-C10 alkyl, or aryl; each being        optionally substituted with 1-4 independent OH, halo, CN, NO₂,        or CO₂H;

wherein R¹ taken together with R² and the nitrogen atom to which theyare attached is not 4-phenyl-piperazin-1-yl,4-(pyridin-4-yl)-piperazin-1-yl, 4-(pyridin-2-yl)-piperazin-1-yl,4-(2-nitro-phenyl)-piperazin-1-yl,4-(3,5-dimethoxy-phenyl)-piperazin-1-yl, or4-[bis-(4-fluoro-phenyl)-methyl]-piperazin-1-yl;

-   -   by reacting an indole of Formula 2:

-   -   wherein each R⁴ and n is independently as defined above;    -   with a reagent of Formula 3:

-   -   wherein X is a leaving group;    -   followed by addition of HNR¹R², where R¹ and R² are as defined        above; and    -   reacting the resulting compound of Formula 4:

-   -   wherein all variables are as defined above;    -   with a base and a compound of the formula XCH₂R³; where X is        independently a leaving group and R³ is as defined above;    -   resulting in a compound of Formula 1.

The invention also relates to a method of making a compound of Formula1:

wherein,

-   -   each R¹ is independently isoxazolyl, thiazolyl, isothiazolyl,        1,3,4-thiadiazolyl, 1,3-benzothiazolyl, quinolyl, isoquinolyl,        thionapthenyl, or, benzofuranyl, each being optionally        substituted with 1-6 independent R⁵; or when taken together with        R² and the nitrogen atom to which they are attached form a 5-8        membered ring comprising C, N, S, or O atoms wherein any atom is        optionally substituted with an independent R⁵;    -   each R² is independently H, C1-C10 alkyl, or aryl, each being        optionally substituted with 1-4 independent R⁵; or when taken        together with R¹ and the nitrogen atom to which they are        attached form a 5-8 membered ring comprising C, N. S, or O atoms        wherein any atom is optionally substituted with an independent        R⁵;    -   each R³ is independently C1-C10 alkyl, C2-C10 alkenyl, C2-C10        alkynyl, C3-C10 cycloalkyl, C4-C10 cycloalkenyl, aryl,        heteroaryl, or heterocyclyl, each being optionally substituted        with 1-4 independent R⁵;    -   each R⁴ is independently H, NO₂, halo, CN, R⁷, OR⁷, CO₂R⁷, SR⁷,        NR⁷R⁷, C(O)R⁷, C(O)NR⁷R⁷, OC(O)R⁷, S(O)₂R⁷, S(O)₂NR⁷R⁷,        NR⁷C(O)NR⁷R⁷, NR⁷C(O)R⁷, NR⁷(COOR⁷), NR⁷S(O)₂NR⁷R⁷, or        NR⁷S(O)₂R⁷, S(O)₂OR⁷;    -   each n is 0, 1, 2, 3, or 4;    -   each R⁵ is independently H, C1-C10 alkyl optionally-substituted        with 1-4 independent R⁶, C2-C10 alkenyl, C2-C10 alkynyl; C3-C10        cycloalkyl; aryl optionally substituted with 1-4 independent R⁶,        heteroaryl optionally substituted with 1-4 independent R⁶,        heterocyclyl optionally substituted with 1-4 independent R⁶,        halo, haloalkyl, SR⁷, OR⁷, NR⁷R⁷, COOR⁷, NO₂, CN, C(O)R⁷,        C(O)NR⁷R⁷, OC(O)R⁷, S(O)₂R⁷, S(O)₂OR⁷, S(O)₂NR⁷R⁷, NR⁷C(O)NR⁷R⁷,        NR⁷C(O)R⁷, NR⁷(COOR⁷), NR⁷S(O)₂NR⁷R⁷, or NR⁷S(O)₂R⁷;    -   each R⁶ is independently C1-C10 alkyl, C2-C10 alkenyl, C2-C10        alkynyl, halo, haloalkyl, CN, NO₂, OR⁷, or SO₂R⁷;    -   each R⁷ is independently H, C1-C10 alkyl, C2-C10 alkenyl, C2-C10        alkynyl; C3-C10 cycloalkyl; aryl, heteroaryl, or heterocyclyl,        each optionally substituted with 14 independent R⁸;    -   each R⁸ is independently H, OH, OR⁹, C1-C10 alkyl, halo, aryl,        NO₂, or CN; and    -   each R⁹ is independently H, C1-C10 alkyl, or aryl; each being        optionally substituted with 14 independent OH, halo, CN, NO₂, or        CO₂H;

wherein R¹ taken together with R² and the nitrogen atom to which theyare attached is not 4-phenyl-piperazin-1-yl,4-(pyridin-4-yl)-piperazin-1-yl, 4-(pyridin-2-yl)-piperazin-1-yl,4-(2-nitro-phenyl)-piperazin-1-yl,4-(3,5-dimethoxy-phenyl)-piperazin-1-yl, or4-[bis-(4-fluoro-phenyl)-methyl]-piperazin-1-yl;

-   -   including reacting an indole of Formula 2:

-   -   wherein each R⁴ and n is independently defined as above;    -   with a base, and a compound of the formula XCH₂R³, wherein X is        independently a leaving group and R³ is as defined above; and    -   reacting the resulting compound of Formula 3:

where R³, R⁴ and n are as defined above;

with a reagent of Formula 4:

-   -   wherein X is independently a leaving group;

and a reagent HNR¹R²; where R¹ and R² are as defined above;

-   -   resulting in a compound of Formula 1.

Another aspect of the invention includes a method of making a compoundof Formula 1

wherein,

-   -   each R¹ is independently C1-C10 alkyl, C2-C10 alkenyl, C2-C10        alkynyl, C3-C10 cycloakyl, C4-C10 cycloalkenyl, aryl,        heteroaryl, or heterocyclyl, each being optionally substituted        with 1-4 independent R⁵; or when taken together with R² and the        nitrogen atom to which they are attached form a 5-8 membered        ring comprising C, N, S, or O atoms wherein any atom is        optionally substituted with an independent R⁵;    -   each R² is independently H, C1-C10 alkyl, or aryl, each being        optionally substituted with 1-4 independent R⁵; or when taken        together with R¹ and the nitrogen atom to which they are        attached form a 5-8 membered ring comprising C, N, S, or O atoms        wherein any atom is optionally substituted with an independent        R⁵;    -   each R³ is independently cyanophenyl, furanyl, thiophenyl,        isoxazolyl, thiazolyl, imidazolyl, 4,5-dihydro-5-isoxazolyl, or        naphthyl; each being optionally substituted with 1-4 independent        R⁵;    -   each R⁴ is independently H, NO₂, halo, CN, R⁷, OR⁷, CO₂R⁷, SR⁷,        NR⁷R⁷, C(O)R⁷, C(O)NR⁷R⁷, OC(O)R⁷, S(O)₂R⁷, S(O)₂NR⁷R⁷,        NR⁷C(O)NR⁷R⁷, NR⁷C(O)R⁷, NR⁷(COOR⁷), NR⁷S(O)₂NR⁷R⁷, or        NR⁷S(O)₂R⁷, S(O)₂OR⁷;    -   each n is 0, 1, 2, 3, or 4;    -   each R⁵ is independently H, C1-C10 alkyl optionally substituted        with 1-4 independent R⁶, C2-C10 alkenyl, C2-C10 alkynyl; C3-C10        cycloalkyl; aryl optionally substituted with 1-4 independent R⁶,        heteroaryl optionally substituted with 1-4 independent R⁶,        heterocyclyl optionally substituted with 14 independent R⁶,        halo, haloalkyl, SR⁷, OR⁷, NR⁷R⁷, COOR⁷, NO₂, CN, C(O)R⁷,        C(O)NR⁷R⁷, OC(O)R⁷, S(O)₂R⁷, S(O)₂OR⁷, S(O)₂NR⁷R⁷, NR⁷C(O)NR⁷R⁷,        NR⁷C(O)R⁷, NR⁷(COOR⁷), NR⁷S(O)₂NR⁷R⁷, or NR⁷S(O)₂R⁷;    -   each R⁶ is independently C1-C10 alkyl, C2-C10 alkenyl, C2-C10        alkynyl, halo, haloalkyl, CN, NO₂, OR⁷, or SO₂R⁷;    -   each R⁷ is independently H, C1-C10 alkyl, C2-C10 alkenyl, C2-C10        alkynyl; C3-C10 cycloalkyl; aryl, heteroaryl, or heterocyclyl,        each optionally substituted with 14 independent R⁸;    -   each R⁸ is independently H, OH, OR⁹, C1-C10 alkyl, halo, aryl,        NO₂, or CN; and    -   each R⁹ is independently H, C1-C10 alkyl, or aryl; each being        optionally substituted with 1-4 independent OH, halo, CN, NO₂,        or CO₂H;

wherein R¹ taken together with R² and the nitrogen atom to which theyare attached is not 4-phenyl-piperazin-1-yl,4-(pyridin-4-yl)-piperazin-1-yl, 4-(pyridin-2-yl)-piperazin-1-yl,4-(2-nitro-phenyl)-piperazin-1-yl,4-(3,5-dimethoxy-phenyl)-piperazin-1-yl, or4-[bis-(4-fluoro-phenyl)-methyl]-piperazin-1-yl;

-   -   including reacting an indole of Formula 2:

-   -   wherein each R⁴ and n is independently as defined above;    -   with a base, and a compound of the formula XCH₂R³, wherein X is        independently a leaving group and R³ is as defined above;    -   reacting the resulting compound of Formula 3:

where R³, R⁴ and n are independently as defined above;

with a reagent having Formula 4:

wherein X is independently a leaving group; and

a reagent HNR¹R²; where R¹ and R² are as defined above;

-   -   resulting in a compound of Formula 1.

The invention also relates to a method of making a composition includingcombining a compound of any of the formulae herein with apharmaceutically acceptable carrier.

Additionally, another aspect of the invention is a method of reducingtumor size including administering a compound of any of the formulaeherein.

Thus, one aspect of the invention relates to a method of making acompound of the formulae described herein, including synthesizing anyone or more intermediates illustrated in the synthetic schemes hereinand then converting that intermediate(s) to a compound of the formulaedescribed herein. Another embodiment relates to a method of making acompound of the formulae described herein, including synthesizing anyone or more intermediates illustrated in the examples herein and thenconverting that intermediate(s) to a compound of the formulae describedherein. Another embodiment relates to a method of making a compound ofthe formulae described herein, including synthesizing any one or moreintermediates illustrated in the synthetic schemes herein and thenconverting that intermediate(s) to a compound of the formulae describedherein utilizing one or more of the chemical reactions described in thesynthetic schemes or examples herein.

The terms “halo” and “halogen” refer to any radical of fluorine,chlorine, bromine or iodine. The terms “alkyl”, “alkenyl” and “alkynyl.”refer to hydrocarbon chains that can be straight-chain orbranched-chain, containing the indicated number of carbon atoms. Forexample, C1-C10 indicates the group can have from 1 to 10 (inclusive)carbon atoms in it. The terms “ring” and “ring system” refer to a ringcomprising the delineated number of atoms, said atoms being carbon or,where indicated, a heteroatom such as nitrogen, oxygen or sulfur. Thering itself, as well as any substituents thereon, can be attached at anyatom that allows a stable compound to be formed.

The term “aryl” refers to a 6-carbon monocyclic or 10-carbon bicyclicaromatic ring system wherein 0, 1, 2 or 3 atoms of each ring can besubstituted by a substituent. Examples of aryl groups include phenyl,naphthyl and the like.

The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring systemcomprising 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic,or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, orS, wherein 0, 1, 2 or 3 atoms of each ring can be substituted by asubstituent. Examples of heteroaryl groups include pyridyl, furyl orfuranyl, imidazolyl, benzimidazolyl, pyrimidinyl, thiophenyl or thienyl,quinolinyl, indolyl, thiazolyl, and the like.

The term “heterocyclyl” refers to a nonaromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring systemcomprising 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic,or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, orS, wherein 0, 1, 2 or 3 atoms of each ring can be substituted by asubstituent. Examples of heterocyclyl groups include piperazinyl,pyrrolidinyl, dioxanyl, morpholinyl, tetrahydrofuranyl, and the like.

The term “multidrug resistant phenotype” refers to cancer cells thatexpress P-glycoprotein (MDR), multidrug resistance-associated proteins(MRP), lung cancer resistance-associated proteins (LRP), breast cancerresistance proteins (BCRP) or other proteins associated with theresistance to anticancer drugs.

The term “treating” or “treated” refers to administering a compounddescribed herein to a subject with the purpose to cure, heal, alleviate,relieve, alter, remedy, ameliorate, improve, or affect a disease, thesymptoms of the disease or the predisposition toward the disease.

“An effective amount” refers to an amount of a compound which confers atherapeutic effect on the treated subject. The therapeutic effect may beobjective (i.e., measurable by some test or marker) or subjective (i.e.,subject gives an indication of or feels and effect). An effective amountof the compound described above may range from about 0.1 mg/Kg to about500 mg/Kg, alternatively from about 1 to about 50 mg/Kg. Effective doseswill also vary depending on route of administration, as well as thepossibility of co-usage with other agents.

Combinations of substituents and variables envisioned by this inventionare only those that result in the formation of stable compounds. Theterm “stable”, as used herein, refers to compounds which possessstability sufficient to allow manufacture and which maintains theintegrity of the compound for a sufficient period of time to be usefulfor the purposes detailed herein (e.g., therapeutic administration to asubject for the treatment of cancer).

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,suitable methods and materials are described below. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

The invention can provide several advantages over the existing methodsof treatment. For example, the compounds of the invention can haveseveral chemical and pharmacological advantages useful in treatingcancer and inhibiting angiogenesis. These advantages can include bothchemical stability and pharmacological stability, as well as potency,different resistance profiles, different selectivity profiles, anddecreased side-effects. The methods and compositions are orally activeagainst cancer and are capable of inhibiting angiogenesis. A simple andefficacious method of treatment for the invention can be provided byeasy administrations through the oral route, which could be distributedin a form that the patient could self-administer repeatedly for multipledoses by which an angiogenesis inhibitor is preferentially administered,sometimes for a long-term regimen for the purpose of disease prevention.The invention also envisions veterinary uses for the treatment of cancerand inhibition of angiogenesis in animals (e.g., dogs, cats, or horses).Thus, a subject as described herein includes these animals as well ashumans.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DESCRIPTION OF THE FIGURES

FIG. 1 is a graph of the dose-response relationship of compound 56,showing survival fractions mice.

FIG. 2 is a graph of the dose-response relationship of compound 25,showing survival fractions of mice.

DETAILED DESCRIPTION

The invention also relates to the specific compounds exemplified herein.Thus one embodiment of the invention is any compound specificallydescribed herein, including the compounds listed below:

TABLE 1 Compounds

Compound R¹ R² R³ R⁴ 1 4-pyridyl H 4-cyanophenyl H 25-(tert-butyl)-3-isoxazolyl H 4-cyanophenyl H 3 6-quinolyl H4-cyanophenyl H 4 4-morpholinophenyl H 4-cyanophenyl H 52,6-difluorophenyl H 4-cyanophenyl H 6 2,6-dichlorophenyl H4-cyanophenyl H 7 4-methoxyphenyl H 4-cyanophenyl H 81,4-benzodioxan-6-yl H 4-cyanophenyl H 9 3-(benzyloxy)phenyl H4-cyanophenyl H 10 3,4,5-trimethoxyphenyl H 4-cyanophenyl H 114-bromophenyl H 4-cyanophenyl H 12 5-methyl-1,3-thiazol-2-yl H4-cyanophenyl H 13 4-iodophenyl H 4-cyanophenyl H 14 3,5-dimethylphenylH 4-cyanophenyl H 15 4-carboethoxyphenyl H 4-cyanophenyl H 16 benzyl H4-cyanophenyl H 17 4-nitrobenzyl H 4-cyanophenyl H 181,2,4,5-tetrahydrooxepin-1-yl H 4-cyanophenyl H 19 5-bromo-2-pyridyl H4-cyanophenyl H 20 4-(benzyloxy)phenyl H 4-cyanophenyl H 216-(4-chlorophenyl)hexyl H 4-cyanophenyl H 22 4-(dimethylamino)phenyl H4-cyanophenyl H 23 4-(acetylamino)phenyl H 4-cyanophenyl H 243-methyl-5-isothiazolyl H 4-cyanophenyl 5-Br 25 3-methyl-5-isothiazolylH 4-cyanophenyl H 26 5-methyl-1,3,4-thiadiazol-2-yl H 4-cyanophenyl H 273-methyl-5-isoxazolyl H 4-cyanophenyl H 28 3-(tert-butyl)-5-isoxazolyl H4-cyanophenyl H 29 3-phenyl-5-isoxazolyl H 4-cyanophenyl H 303-(4-chlorophenyl)-5-isoxazolyl H 4-cyanophenyl H 31 4-methoxyphenyl4-cyanobenzyl 4-cyanophenyl H 32 4-methoxyphenyl H 2-cyanophenyl H 334-methoxyphenyl 2-cyanobenzyl 2-cyanophenyl H 344-methyl-1,3-thiazol-2-yl H 4-chlorophenyl H 355-methyl-1,3,4-thiadiazol-2-yl H 4-chlorophenyl H 363-(tert-butyl)-5-isoxazolyl H 4-chlorophenyl H 37 3-phenyl-5-isoxazolylH 4-chlorophenyl H 38 3-(4-chlorophenyl)-5-isoxazolyl H 4-chlorophenyl H39 3-methyl-5-isothiazolyl H 3,4,5-trimethoxyphenyl H 403-methyl-5-isothiazolyl H 2-furyl H 41 4-pyridyl H 2-furyl H 426-quinolyl H 2-furyl H 43 6-(4-chlorophenoxy)hexyl H 4-cyanophenyl H 443-methyl-5-isothiazolyl H 2-thienyl H 45 3-methyl-5-isothiazolyl H3-thienyl H 46 3-methyl-5-isoxazolyl H 3-thienyl H 47 4-pyridyl H3-thienyl H 48 6-quinolyl H 3-thienyl H 49 4-methyl-thiazol-2-yl H3-thienyl H 50 4-methoxyphenyl H 3-thienyl H 51 3-methyl-5-isothiazolylH 3-methyl-4,5-dihydro-5- H isoxazolyl 523-(4-chlorophenyl)-5-isoxazolyl H 3-methyl-4,5-dihydro-5- H isoxazolyl53 4-bromophenyl H 5-isoxazolyl H 54 3-methyl-5-isothiazolyl H5-isoxazolyl H 55 4-pyridyl H 5-isoxazolyl H 56 3-methyl-5-isothiazolylH 3-methyl-5-isoxazolyl H 57 3-methyl-5-isoxazolyl H3-methyl-5-isoxazolyl H 58 4-pyridyl H 3-methyl-5-isoxazolyl H 596-quinolyl H 3-methyl-5-isoxazolyl H 60 4-methyl-1,3-thiazol-2-yl H3-methyl-5-isoxazolyl H 61 1,3-benzothiazol-2-yl H 3-methyl-5-isoxazolylH 62 4-bromophenyl H 3-methyl-5-isoxazolyl H 63 3-methyl-5-isothiazolylH 3-ethyl-5-isoxazolyl H 64 4-chlorophenyl H 3-ethyl-5-isoxazolyl H 653-methyl-5-isothiazolyl H 3-isopropyl-5-isoxazolyl H 665-methyl-3-isoxazolyl H 3-isopropyl-5-isoxazolyl H 67 4-pyridyl H3-isopropyl-5-isoxazolyl H 68 4-methyl-1,3-thiazol-2-yl H3-isopropyl-5-isoxazolyl H 69 4-bromophenyl H 3-isopropyl-5-isoxazolyl H70 3-methyl-5-isothiazolyl H 3-phenyl-5-isoxazolyl H 71 4-pyridyl H3-phenyl-5-isoxazolyl H 72 4-methyl-1,3-thiazol-2-yl H3-phenyl-5-isoxazolyl H 73 4-bromophenyl H 3-phenyl-5-isoxazolyl H 743-(4-chlorophenyl)-5-isoxazolyl H 3-phenyl-5-isoxazolyl H 754-methyl-1,3-thiazol-2-yl H 4-thiazolyl H 76 4-pyridyl H 4-thiazolyl H77 6-quinolyl H 4-thiazolyl H 78 4-bromophenyl H 1-methyl-1H-5- Himidazolyl 79 4-chlorophenyl H 1-methyl-1H-5- H imidazolyl 805-methyl-3-isoxazolyl H 1-methyl-1H-5- H imidazolyl 813-methyl-5-isothiazolyl H 4-pyridyl H 82 4-methyl-1,3-thiazol-2-yl H4-pyridyl H 83 5-methyl-3-isoxazolyl H 4-pyridyl H 843-(tert-butyl)-5-isoxazolyl H 4-pyridyl H 85 3-methyl-5-isothiazolyl H3-pyridyl H 86 3-methyl-5-isoxazolyl H 3-pyridyl H 874-methyl-1,3-thiazol-2-yl H 3-pyridyl H 88 5-methyl-3-isoxazolyl H3-pyridyl H 89 5-(tert-butyl)-3-isoxazolyl H 3-pyridyl H 905-methyl-3-isoxazolyl H 4-methoxyphenyl H 91 5-methyl-3-isoxazolyl H3,4-difluorophenyl H 92 5-methyl-3-isoxazolyl H phenyl H 935-methyl-3-isoxazolyl H 2-naphthyl H 94 5-methyl-3-isoxazolyl H3-phenoxypropyl H 95 5-methyl-3-isoxazolyl H 2-fluorophenyl H 965-methyl-3-isoxazolyl H 3-methyl-2-butenyl H 97 5-methyl-3-isoxazolyl Hisopropenyl H 98 5-methyl-3-isoxazolyl H 2-methyl-1-propenyl H 995-[(4-nitrophenyl)sulfonyl]-1,3- H dimethyl H thiazol-2-yl 1003-methyl-5-isothiazolyl H 4-cyanophenyl 5-methoxy 1015-methyl-3-isoxazolyl H 3,4,5-trimethoxybenzyl 5-cyano 1023-methyl-5-isothiazolyl H 4-cyanophenyl 5-intro

The compounds of this invention can be synthesized using conventionaltechniques. Advantageously, these compounds are conveniently synthesizedfrom readily available starting materials. In general, the compounds ofthe formulae described herein are conveniently obtained via standardorganic chemistry synthesis methods, including those methods illustratedin the schemes and the examples herein.

Nucleophilic agents are known in the art and are described in thechemical texts and treatises referred to herein, and include reagentshaving electrons to share. Leaving groups are known in the art and areany stable species that can be detached from a molecule during areaction (e.g., halides, triflates, alkoxides, or alcohols). A base isknown in the art and is any species that has a pair of electronsavailable to share with a proton (eg., primary and secondary amines,tert-butoxides, pyridine, or hydrides). The chemicals used in theaforementioned methods can include, for example, solvents, reagents,catalysts, protecting group and deprotecting group reagents and thelike. The methods described above can also additionally include steps,either before or after the steps described specifically herein, to addor remove suitable protecting groups in order to ultimately allowsynthesis of the compound of the formulae described herein.

As can be appreciated by the skilled artisan, the synthetic schemesherein are not intended to constitute a comprehensive list of all meansby which the compounds described and claimed in this application can besynthesized. Further methods will be evident to those of ordinary skillin the art. Additionally, the various synthetic steps described abovecan be performed in an alternate sequence or order to give the desiredcompounds. Synthetic chemistry transformations and protecting groupmethodologies (protection and deprotection) useful in synthesizing thecompounds described herein are known in the art and include, forexample, those such as described in R. Larock, Comprehensive OrganicTransformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts,Protective Groups in Organic Synthesis, 2nd. Ed., John Wiley and Sons(1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents forOrganic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed.,Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons(1995); and subsequent editions thereof.

In general, the indole derivatives of the present invention are preparedaccording to the synthetic scheme shown below. Variables and groups inthe chemical structural formulas in the methods below are defined asdelineated herein for any of the formulae, including formula 1.

A solution of indolyl compound starting material in solvent (e.g.,tetrahydrofuran, isopropanol, dichloromethane, dioxane, dimethylformamide, dimethyl sulfoxide, or toluene) is reacted with a base (e.g.,sodium hydride, potassium hydroxide, or potassium tert-butoxide) and acompound of the formula XCH₂R³ (where X is a leaving group). Theresulting intermediate is reacted with an oxalyl derivative and an amineof the formula MNR¹R² (where M is H or metal cation, e.g., K, Li, Na),to give compounds of the formulae delineated herein. The desiredcompounds or intermediates can be isolated and purified using standardsynthetic techniques or can be reacted further (i.e., “one-potsynthesis”) without isolation or purification.

Alternatively, the indolyl compounds of the present invention areprepared according to the following synthetic scheme shown below.

The indolyl starting material is dissolved in solvent (e.g., diethylether, tetrahydrofuran, or dichloromethane) and reacted with an oxalylderivative (e.g., oxalyl chloride) and an amine of the formula MNR¹R²(where M is H or metal cation, e.g., K, Li, Na). The intermediate iscompound is reacted with a compound of the formula XCH₂R³ (where X is aleaving group) to give compounds of the formulae herein. The desiredcompounds or intermediates can be isolated (and optionally purified) orcan be reacted further (i.e., “one-pot synthesis” without isolation orpurification).

As used herein, the compounds of this invention, including the compoundsof formulae described herein, are defined to include pharmaceuticallyacceptable derivatives or prodrugs thereof. A “pharmaceuticallyacceptable derivative or prodrug” means any pharmaceutically acceptablesalt, ester, salt of an ester, or other derivative of a compound of thisinvention which, upon administration to a recipient, is capable ofproviding (directly or indirectly) a compound of this invention.Particularly favored derivatives and prodrugs are those that increasethe bioavailability of the compounds of this invention when suchcompounds are administered to a subject (e.g., by allowing an orallyadministered compound to be more readily absorbed into the blood) orwhich enhance delivery of the parent compound to a biologicalcompartment (e.g., the brain or lymphatic system) relative to the parentspecies. Preferred prodrugs include derivatives where a group whichenhances aqueous solubility or active transport through the gut membraneis appended to the structure of formulae described herein.

The compounds of this invention can be modified by appending appropriatefunctionalities to enhance selective biological properties. Suchmodifications are known in the art and include those which increasebiological penetration into a given biological compartment (e.g., blood,lymphatic system, central nervous system), increase oralbioavailability, increase solubility to allow administration byinjection, alter metabolism and alter rate of excretion.

Pharmaceutically acceptable salts of the compounds of this inventioninclude those derived from pharmaceutically acceptable inorganic andorganic acids and bases. Examples of suitable acid salts includeacetate, adipate, alginate, aspartate, benzoate, benzenesulfonate,butyrate, citrate, camphorate, camphorsulfonate, ethanesulfonate,formate, fumarate, glycolate, heptanoate, hexanoate, hydrochloride,hydrobromide, 2-hydroxyethanesulfonate, lactate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, palmoate,pectinate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate,salicylate, succinate, sulfate, tartrate, thiocyanate, tosylate andundecanoate. Other acids, such as oxalic, while not in themselvespharmaceutically acceptable, can be employed in the preparation of saltsuseful as intermediates in obtaining the compounds of the invention andtheir pharmaceutically acceptable acid addition salts. Salts derivedfrom appropriate bases include alkali metal (e.g., sodium), alkalineearth metal (e.g., magnesium), ammonium and N-(alkyl)₄ ⁺ salts. Thisinvention also envisions the quaternization of any basicnitrogen-containing groups of the compounds disclosed herein. Water oroil-soluble or dispersible products can be obtained by suchquaternization.

The anticancer, antitumor, cytotoxicity, antiangiogenic or otherbiological activity of the compounds can be assayed by standard methodsand assays known in the art, including those exemplified by the examplesdescribed herein. These analyses are useful for assessing anddemonstrating the efficacy of the compounds herein as anticancer,antitumor, or cytotoxic agents.

The heterocyclic compounds of the formulae delineated herein can beadministered to a patient, for example, in order to treat cancer or toprevent unwanted angiogenesis. The heterocyclic compounds can, forexample, be administered in a pharmaceutically acceptable carrier suchas physiological saline, in combination with other drugs, and/ortogether with appropriate excipients.

As the skilled artisan will appreciate, lower or higher doses than thoserecited above can be required. Specific dosage and treatment regimensfor any particular patient will depend upon a variety of factors,including the activity of the specific compound employed, the age, bodyweight, general health status, sex, diet, time of administration, routeof administration, frequency of administration, rate of excretion, drugcombination, the severity and course of the disease, condition orsymptoms, the patient's disposition to the disease, condition orsymptoms, and the judgment of the treating physician.

In the methods of treating, preventing, or relieving symptoms ofdiseases (e.g., cancer, tumors, proliferation of new blood vessels) in amammal including any of the pharmaceutical compositions and combinationsdescribed above, preferably the mammal is a human. If the pharmaceuticalcomposition includes only the compound of this invention as the activecomponent, such methods can additionally include administering to saidmammal an additional therapeutic agent such as, for example, paclitaxel,docitaxel, doxorubicin, daunorubicin, epirubicin, fluorouracil,melphalan, cis-platin, carboplatin, cyclophosphamide, mitomycin,methotrexate, mitoxantrone, vinblastine, vincristine, ifosfamide,teniposide, etoposide, bleomycin, leucovorin, cytarabine, dactinomycin,interferon alpha, streptozocin, prednisolone, procarbazine, SU5416,SU6668, endostatin, angiostatin, combretastatin A4-phosphate,thalidomide, 2-methoxyestradiol, CAI, CC-5013, LY317615. Other suitableagents are delineated in texts and publications, including for exampleCancer: Principles & Practice of Oncology, 6th Edition by Vincent T.DeVita, Jr., Samuel Hellman, and Steven A. Rosenberg, Lippincott-RavenPublishers, Philadelphia, USA, 2001. Such additional agent(s) can beadministered to the mammal prior to, concurrently with, or following theadministration of the composition having a compound of any of theformulae herein.

Pharmaceutical compositions of this invention include a compound of theformulae described herein or a pharmaceutically acceptable salt thereof;an additional agent, such as an anticancer agent, an anti-angiogenicagent (e.g., protamine, heparin, interferons, steroids, DS 4152, AGM12470, SU5416, SU6668, combretastatin A4-phosphate, angiostatin,endostatin, TNP-470, 2-methoxyestradiol, thalidomide, CAI, CC-5013,LY317615.), and any pharmaceutically acceptable carrier, adjuvant orvehicle. Alternate compositions of this invention include a compound ofthe formulae described herein or a pharmaceutically acceptable saltthereof; and a pharmaceutically acceptable carrier, adjuvant or vehicle.Such compositions can optionally include additional therapeutic agents,including, for example an additional agent selected from an anticanceragent, an anti-angiogenic agent, an antiviral agent, an antibiotic, apain relief agent, an antianemia agent (e.g., erythropoetin), a cytokine(e.g., granulocyte-macrophage colony-stimulating factor, granulocytecolony-stimulating factor, interleukins) or an antinausea agent. Thecompositions delineated herein include the compounds of the formulaedelineated herein, as well as additional therapeutic agents if present,in amounts effective for achieving a modulation of the levels or thepresence of cancerous cells.

The term “pharmaceutically acceptable carrier or adjuvant” refers to acarrier or adjuvant that can be administered to a patient, together witha compound of this invention, and which does not destroy thepharmacological activity thereof and is nontoxic when administered indoses sufficient to deliver a therapeutic amount of the compound.

Pharmaceutically acceptable carriers, adjuvants and vehicles that can beused in the pharmaceutical compositions of this invention include, butare not limited to, ion exchangers, alumina, aluminum stearate,lecithin, self-emulsifying drug delivery systems (SEDDS) such asd-α-tocopherol polyethylene glycol 1000 succinate, surfactants used inpharmaceutical dosage forms such as Tweens or other similar polymericdelivery matrices, serum proteins, such as human serum albumin, buffersubstances such as phosphates, glycine, sorbic acid, potassium sorbate,partial glyceride mixtures of saturated vegetable fatty acids, water,salts or electrolytes, such as protamine sulfate, disodium hydrogenphosphate, potassium hydrogen phosphate, sodium chloride, zinc salts,colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone,cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-blockpolymers, and wool fat. Cyclodextrins such as α-, β-, andγ-cyclodextrin, or chemically modified derivatives such ashydroxyalkylcyclodextrins, including 2- and3-hydroxypropyl-β-cyclodextrins, or other solubilized derivatives canalso be advantageously used to enhance delivery of compounds of theformulae described herein.

The pharmaceutical compositions of this invention can be administeredorally, parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally, subdermally, transmucosally, or via an implantedreservoir, preferably by oral administration or administration byinjection. The pharmaceutical compositions of this invention can containany conventional non-toxic pharmaceutically-acceptable carriers,adjuvants or vehicles. In some cases, the pH of the formulation can beadjusted with pharmaceutically acceptable acids, bases or buffers toenhance the stability of the formulated compound or its delivery form.The term parenteral as used herein includes subcutaneous,intracutaneous, intravenous, intramuscular, intraarticular,intraarterial, intrasynovial, intrasternal, intrathecal, intralesional,intraperitoneally, and intracranial injection or infusion techniques.

The pharmaceutical compositions can be in the form of a sterileinjectable preparation, for example, as a sterile injectable aqueous oroleaginous suspension. This suspension can be formulated according totechniques known in the art using suitable dispersing or wetting agents(such as, for example, Tween 80) and suspending agents. The sterileinjectable preparation can also be a sterile injectable solution orsuspension in a non-toxic parenterally acceptable diluent or solvent,for example, as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that can be employed are mannitol, water, Ringer'ssolution and isotonic sodium chloride solution. In addition, sterile,fixed oils are conventionally employed as a solvent or suspendingmedium. For this purpose, any bland fixed oil can be employed includingsynthetic mono- or di-glycerides. Fatty acids, such as oleic acid andits glyceride derivatives are useful in the preparation of injectables,as are natural pharmaceutically-acceptable oils, such as olive oil orcastor oil, especially in their polyoxyethylated versions. These oilsolutions or suspensions can also contain a long-chain alcohol diluentor dispersant, or carboxymethyl cellulose or similar dispersing agentswhich are commonly used in the formulation of pharmaceuticallyacceptable dosage forms such as emulsions and/or suspensions. Othercommonly used surfactants such as Tweens or Spans and/or other similaremulsifying agents or bioavailability enhancers which are commonly usedin the manufacture of pharmaceutically acceptable solid, liquid, orother dosage forms can also be used for the purposes of formulation.

The pharmaceutical compositions of this invention can be orallyadministered in any orally acceptable dosage form including, but notlimited to, capsules, tablets, emulsions and aqueous suspensions,dispersions and solutions. In the case of tablets for oral use, carrierswhich are commonly used include lactose and corn starch. Lubricatingagents, such as magnesium stearate, are also typically added. For oraladministration in a capsule form, useful diluents include lactose anddried corn starch. When aqueous suspensions and/or emulsions areadministered orally, the active ingredient can be suspended or dissolvedin an oily phase combined with emulsifying and/or suspending agents. Ifdesired, certain sweetening and/or flavoring and/or coloring agents canbe added.

The pharmaceutical compositions of this invention can also beadministered in the form of suppositories for rectal administration.These compositions can be prepared by mixing a compound of thisinvention with a suitable non-irritating excipient which is solid atroom temperature but liquid at the rectal temperature and therefore willmelt in the rectum to release the active components. Such materialsinclude, but are not limited to, cocoa butter, bees wax and polyethyleneglycols.

Topical administration of the pharmaceutical compositions of thisinvention is especially useful when the desired treatment involves areasor organs readily accessible by topical application. For applicationtopically to the skin, the pharmaceutical composition should beformulated with a suitable ointment containing the active componentssuspended or dissolved in a carrier. Carriers for topical administrationof the compounds of this invention include, but are not limited to,mineral oil, liquid petroleum, white petroleum, propylene glycol,polyoxyethylene polyoxypropylene compound, emulsifying wax and water.Alternatively, the pharmaceutical composition can be formulated with asuitable lotion or cream containing the active compound suspended ordissolved in a carrier with suitable emulsifying agents. Suitablecarriers include, but are not limited to, mineral oil, sorbitanmonostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol,2-octyldodecanol, benzyl alcohol and water. The pharmaceuticalcompositions of this invention can also be topically applied to thelower intestinal tract by rectal suppository formulation or in asuitable enema formulation. Topically applied transdermal patches arealso included in this invention.

The pharmaceutical compositions of this invention can be administered bynasal aerosol or inhalation. Such compositions are prepared according totechniques well-known in the art of pharmaceutical formulation and canbe prepared as solutions in saline, employing benzyl alcohol or othersuitable preservatives, absorption promoters to enhance bioavailability,fluorocarbons, and/or other solubilizing or dispersing agents known inthe art.

Dosage levels of between about 0.001 and about 100 mg/kg body weight perday, alternatively between about 0.5 and about 75 mg/kg body weight perday of the anticancer compounds described herein are useful in amonotherapy and/or in combination therapy for the prevention andtreatment of cancer. Typically, the pharmaceutical compositions of thisinvention will be administered from about 1 to about 6 times per day(e.g., at 10 mg-100 mg/dose) or alternatively, as a continuous infusion.Such administration can be used as a chronic or acute therapy. Theamount of active ingredient that can be combined with the carriermaterials to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration. A typicalpreparation will contain from about 5% to about 95% active compound(w/w). Alternatively, such preparations contain from about 20% to about80% active compound.

When the compositions of this invention include a combination of acompound of the formulae described herein and one or more additionaltherapeutic or prophylactic agents, both the compound and the additionalagent should be present at dosage levels of between about 10 to 100%,and more preferably between about 10 to 80% of the dosage normallyadministered in a monotherapy regimen. The additional agents can beadministered separately, as part of a multiple dose regimen, from thecompounds of this invention. Alternatively, those agents can be part ofa single dosage form, mixed together with the compounds of thisinvention in a single composition.

The compounds of this invention can contain one or more asymmetriccenters and thus occur as racemates and racemic mixtures, singleenantiomers, individual diastereomers and diastereomeric mixtures. Allsuch isomeric forms of these compounds are expressly included in thepresent invention. The compounds of this invention can also berepresented in multiple tautomeric forms (see illustration), in suchinstances, the invention expressly includes all tautomeric forms of thecompounds described herein. All such isomeric forms of such compoundsare expressly included in the present invention (e.g., alkylation of aring system can result in alkylation at multiple sites, the inventionexpressly includes all such reaction products). All crystal forms of thecompounds described herein are expressly included in the presentinvention.

Substituents on ring moieties (e.g., phenyl, thienyl, etc.) can beattached to specific atoms, whereby they are intended to be fixed tothat atom, or they can be drawn unattached to a specific atom (seebelow), whereby they are intended to be attached at any available atomthat is not already substituted by an atom other than H (hydrogen). Forexample, a structure drawn as:

is intended to encompass all of (but is not limited to) the followingstructures:

All references cited herein, whether in print, electronic, computerreadable storage media or other form, are expressly incorporated byreference in their entirety, including but not limited to, abstracts,articles, journals, publications, texts, treatises, internet web sites,databases, patents, and patent publications.

The invention will be further described in the following example. Itshould be understood that these examples are for illustrative purposesonly and are not to be construed as limiting this invention in anymanner.

EXAMPLES Example 1a Synthesis of Compound 1

A solution of indole (1.17 g, 10 mmol) in 10 mL tetrahydrofuran wasadded dropwise to a suspension of potassium tert-butoxide (1.34 g, 12mmol) in 10 mL tetrahydrofuran. The reaction mixture was stirred at roomtemperature for 2 hours, then α-bromo-p-tolunitrile (1.96 g, 10 mmol) in5 mL tetrahydrofuran was added dropwise. The solution was allowed standfor 4 hours and then 10 mL saturated ammonium chloride was added withstirring. The mixture was extracted three times with a total of 60 mL ofether, the organic phase was dried using anhydrous magnesium sulfate andfiltered, and the filtrate was concentrated in vacuum and purified byflash chromatography on silica gel. The eluent used was a mixture ofn-hexane and ethyl acetate in the ratio 6:1 (vol/vol). Yield: 1.97 g,85%.

A solution of 4-(1H-1-indolylmethyl)benzonitrile (232 mg, 1.0 mmol) in10 mL diethyl ether was added oxalyl chloride (254 mg, 2.0 mmol)dropwise at 0° C. The reaction mixture was stirred at 0° C. for 3 hoursand then the reaction solvent was evaporated. The residue was dissolvedin 5 mL tetrahydrofuran and then the 4-aminopyridine (94 mg, 1.0 mmol)in 10 mL tetrahydrofuran was added dropwise. The mixture was stirred for10 hours and then 1 N NaOH (4 mL) was added to the reaction flaskdropwise. The mixture was extracted three times with a total of 60 mL oftetrahydrofuran, the organic phase was dried using anhydrous magnesiumsulfate and filtered; and the filtrate was concentrated in vacuum. Theresidue was crystallized from methanol. Yield: 0.21 g, 56%.

NMR: 9.51 (s, 1H), 9.12 (s, 1H), 8.61-8.59 (m, 2H), 8.48 (d, J=6.0 Hz,2H), 7.66-7.63 (m, 4H), 7.44-7.23 (m, 5H), 5.50 (s, 2H).

MS (M+1): 381.

Example 1b Synthesis of Compound 1

A solution of indole (1.17 g, 10 mmol) in 10 mL diethyl ether was addedto oxalyl chloride (2.54 g, 20 mmol) dropwise at 0° C. The reactionmixture was stirred at 0° C. for 3 hours and then the reaction solventwas evaporated. The residue was dissolved in 5 mL tetrahydrofuran andthen the 4-aminopyridine (94 mg, 1.0 mmol) in 10 mL tetrahydrofuran wasadded dropwise. The mixture was stirred for 10 hours and then 1 N NaOH(4 mL) was added to the reaction flask dropwise. The mixture wasextracted three times with a total of 60 mL of tetrahydrofuran. Theorganic phase was dried using anhydrous magnesium sulfate and filtered,and the filtrate was concentrated in vacuum. The residue wascrystallized from methanol. Yield: 1.04 g, 63%.

A solution of N1-(4-pyridyl)-2-(1H-3-indolyl)-2-oxoacetamide (825 mg, 5mmol) in 10 mL tetrahydrofuran was added dropwise to a suspension ofpotassium tert-butoxide (0.67 g, 6 mmol) in 10 mL tetrahydrofuran. Thereaction mixture was stirred at room temperature for 2 hours, thenα-bromo tolunitrile (0.98 g, 5 mmol) in 5 mL tetrahydrofuran was addeddropwise. The solution was allowed stand for 4 hours and then 10 mLsaturated ammonium chloride was added with stirring. The mixture wasextracted three times with a total of 60 mL of tetrahydrofuran and theorganic phase was dried using anhydrous magnesium sulfate and filtered.The filtrate was concentrated in vacuum and purified by crystallizedfrom methanol. Yield: 0.99 g, 52%.

NMR: 9.51 (s, 1H), 9.12 (s, 1H), 8.61-8.59 (m, 2H), 8.48 (d, J=6.0 Hz,2H), 7.66-7.63 (m, 4H), 7.44-7.23 (m, 5H), 5.50 (s, 1H).

MS (M+1): 381.

Example 2 Synthesis of Compound 25

A solution of indole (1.17 g, 10 mmol) in 10 mL tetrahydrofuran wasadded dropwise to a suspension of potassium tert-butoxide (1.34 g, 12mmol) in 10 mL tetrahydrofuran. The reaction mixture was stirred at roomtemperature for 2 hours, then α-bromo-ptolunitrile (1.96 g, 10 mmol) in5 mL tetrahydrofuran was added dropwise. The solution was allowed standfor 4 hours and then 10 mL saturated ammonium chloride was added withstirring. The mixture was extracted three times with a total of 60 mL ofether, the organic phase was dried using anhydrous magnesium sulfate andfiltered, and the filtrate was concentrated in vacuum and purified byflash chromatography on silica gel. The eluent was used a mixture ofn-hexane and ethyl acetate in the ratio 6:1 (vol/vol). Yield: 1.97 g,85%.

A solution of 4-(1H-1-indolylmethyl)benzonitrile (232 mg, 1.0 mmol) in10 mL diethyl ether was added to oxalyl chloride (254 mg, 2.0 mmol)dropwise at 0° C. The reaction mixture was stirred at 0° C. for 3 hoursand then the reaction solvent was evaporated. The residue was dissolvedin 5 mL tetrahydrofuran and then the 2-amino-4-methylisothiazolehydrochloride (151 mg, 1.0 mmol) and triethylamine (1 mL) in 10 mLtetrahydrofuran was added dropwise. The mixture was stirred for 10 hoursand then 1 N NaOH (4 mL) was added to the reaction flask dropwise. Themixture was extracted three times with a total of 60 mL oftetrahydrofuran, the organic phase was dried using anhydrous magnesiumsulfate and filtered, and the filtrate was concentrated in vacuum. Theresidue was recrystallized from methanol. Yield: 0.19 g, 48%.

NMR: 10.32 (s, 1H), 9.17 (s, 1H), 8.47 (d, J=7.5 Hz, 1H), 7.65-7.22 (m,7H), 6.82 (s, 1H), 5.49 (s, 2H), 2.47 (s, 3H).

MS (M+1): 401.2.

Example 3 Synthesis of Compound 42

A solution of indole (1.17 g, 10 mmol) in 10 mL tetrahydrofuran wasadded dropwise to a suspension of potassium tert-butoxide (1.34 g, 12mmol) in 10 mL tetrahydrofuran. The reaction mixture was stirred at roomtemperature for 2 hours, then 2-(bromomethyl)furan (1.10 g, 10 mmol) in5 mL tetrahydrofuran was added dropwise. The solution was allowed standfor 4 hours and then 10 mL saturated ammonium chloride was added withstirring. The mixture was extracted three times with a total of 60 mL ofether, the organic phase was dried using anhydrous magnesium sulfate andfiltered, and the filtrate was concentrated in vacuum and purified byflash chromatography on silica gel. The eluent used was a mixture ofn-hexane and ethyl acetate in the ratio 8:1 (vol/vol). Yield: 1.42 g,85%.

A solution of 1-(2-furylmethyl)-1H-indole (197 mg, 1.0 mmol) in 10 mLdiethyl ether was added to oxalyl chloride (254 mg, 2.0 mmol) dropwiseat 0° C. The reaction mixture was stirred at 0° C. for 3 hours and thenthe reaction solvent was evaporated. The residue was dissolved in 5 mLtetrahydrofuran and then the 6-aminoquinoline (144 mg, 1.0 mmol) in 10mL tetrahydrofuran was added dropwise. The mixture was stirred for 10hours and then 1 N NaOH (4 mL) was added to the reaction flask dropwise.The mixture was extracted three times with a total of 60 mL oftetrahydrofuran, the organic phase was dried using anhydrous magnesiumsulfate and filtered, and the filtrate was concentrated in vacuum. Theresidue was crystallized from methanol. Yield: 0.29 g, 74%.

NMR: 9.56 (s, 1H), 9.10 (s, 1H), 8.81 (d, J=3.9 Hz, 1H), 8.45-8.40 (m,2H), 8.12 (d, J=8.7 Hz, 1H), 8.06 (d, J=8.7 Hz, 1H), 7.73 (dd, J=9.0 Hz,J=2.4 Hz, 1H), 7.48-7.31 (m, 4H), 7.19 (s, 1H), 6.35-6.29 (m, 2H), 5.30(s, 2H).

MS (M+1): 396.1

Example 4 Synthesis of Compound 46

A solution of indole (1.17 g, 10 mmol) in 10 mL tetrahydrofuran wasadded dropwise to a suspension of potassium tert-butoxide (1.34 g, 12mmol) in 10 mL tetrahydrofuran. The reaction mixture was stirred at roomtemperature for 2 hours, then 3-(chloromethyl)thiophene (1.33 g, 10mmol) in 5 mL tetrahydrofuran was added dropwise. The solution wasallowed stand for 4 hours and then 10 mL saturated ammonium chloride wasadded with stirring. The mixture was extracted three times with a totalof 60 mL of ether, the organic phase was dried using anhydrous magnesiumsulfate and filtered, and the filtrate was concentrated in vacuum andpurified by flash chromatography on silica gel. The eluent was used amixture of n-hexane and ethyl acetate in the ratio 8:1 (vol/vol). Yield:1.64 g, 77%.

A solution of 1-(3-thienylmethyl)-1H-indole (213 mg, 1.0 mmol) in 10 mLdiethyl ether was added to oxalyl chloride (254 mg, 2.0 mmol) dropwiseat 0° C. The reaction mixture was stirred at 0° C. for 3 hours and thenthe reaction solvent was evaporated. The residue was dissolved in 5 mLtetrahydrofuran and then the 3-methyl-5-isoxazolamine (98 mg, 1.0 mmol)in 10 mL tetrahydrofuran was added dropwise. The mixture was stirred for10 hours and then 1 N NaOH (4 mL) was added to the reaction flaskdropwise. The mixture was extracted three times with a total of 60 mL oftetrahydrofuran, the organic phase was dried using anhydrous magnesiumsulfate and filtered, and the filtrate was concentrated in vacuum. Theresidue was crystallized from methanol. Yield: 0.24 g, 65%.

NMR: 10.07 (s, 1H), 9.00 (s, 1H), 8.47-8.43 (m, 1H), 7.43-7.31 (m, 4H),7.15 (dd, J=2.9 Hz, J=1.2 Hz, 1H), 6.97 (dd, J=5.0 Hz, J=1.2 Hz, 1H),6.35 (s, 1H), 5.41 (s, 2H), 2.32 (s, 3H).

MS (M+1): 366.

Example 5 Synthesis of Compound 56

A solution of indole (1.17 g, 10 mmol) in 10 mL tetrahydrofuran wasadded dropwise to a suspension of potassium tert-butoxide (1.34 g, 12mmol) in 10 mL tetrahydrofuran. The reaction mixture was stirred at roomtemperature for 2 hours, then 5-(chloromethyl)-3-methylisoxazole (1.32g, 10 mmol) in 5 mL tetrahydrofuran was added dropwise. The solution wasallowed stand for 4 hours and then 10 mL saturated ammonium chloride wasadded with stirring. The mixture was extracted three times with a totalof 60 mL of ether, the organic phase was dried using anhydrous magnesiumsulfate and filtered, and the filtrate was concentrated in vacuum andpurified by flash chromatography on silica gel. The eluent used was amixture of n-hexane and ethyl acetate in the ratio 8:1 (vol/vol). Yield:1.61 g, 76%.

A solution of 5-(1H-1-indolylmethyl)-3-methylisoxazole (212 mg, 1.0mmol) in 10 mL diethyl ether was added to oxalyl chloride (254 mg, 2.0mmol) dropwise at 0° C. The reaction mixture was stirred at 0° C. for 3hours and then the reaction solvent was evaporated. The residue wasdissolved in 5 mL tetrahydrofuran and then the3-methyl-5-isothiazolamine (114 mg, 1.0 mmol) and triethylamine (1 mL)in 10 mL tetrahydrofuran was added dropwise. The mixture was stirred for10 hours and then 1 N NaOH (4 mL) was added to the reaction flaskdropwise. The mixture was extracted three times with a total of 60 mL oftetrahydrofuran, the organic phase was dried using anhydrous magnesiumsulfate and filtered, and the filtrate was concentrated in vacuum. Theresidue was crystallized from methanol. Yield: 0.27 g, 71%.

NMR: 10.33 (s, 1H), 9.15 (s, 1H), 8.44 (d, J=6.3 Hz, 1H), 7.45-7.38 (m,3H), 6.82 (s, 1H), 5.96 (s, 1H), 5.48 (s, 2H), 2.49 (s, 3H), 2.52 (s,3H).

MS (M+1): 381.1.

Example 6 Synthesis of Compound 75

A solution of indole (1.17 g, 10 mmol) in 10 mL tetrahydrofuran wasadded dropwise to a suspension of potassium tert-butoxide (1.34 g; 12mmol) in 10 mL tetrahydrofuran. The reaction mixture was stirred at roomtemperature for 2 hours, then 4-(chloromethyl)-1,3-thiazole (1.34 g, 10mmol) in 5 mL tetrahydrofuran was added dropwise. The solution wasallowed stand for 4 hours and then 10 mL saturated ammonium chloride wasadded with stirring. The mixture was extracted three times with a totalof 60 mL of ether, the organic phase was dried using anhydrous magnesiumsulfate and filtered, and the filtrate was concentrated in vacuum andpurified by flash chromatography on silica gel. The eluent used was amixture of n-hexane and ethyl acetate in the ratio 8:1 (vol/vol). Yield:1.43 g, 67%.

A solution of 4-(1H-1-indolylmethyl)-1,3-thiazole (214 mg, 1.0 mmol) in10 mL diethyl ether was added to oxalyl chloride (254 mg, 2.0 mmol)dropwise at 0° C. The reaction mixture was stirred at 0° C. for 3 hoursand then the reaction solvent was evaporated. The residue was dissolvedin 5 mL tetrahydrofuran and then the 4-methyl-1,3-thiazol-2-7 amine (114mg, 1.0 mmol) and triethylamine (1 mL) in 10 mL tetrahydrofuran wasadded dropwise. The mixture was stirred for 10 hours and then 1 N NaOH(4 mL) was added to the reaction flask dropwise. The mixture wasextracted three times with a total of 60 mL of tetrahydrofuran, theorganic phase was dried using anhydrous magnesium sulfate and filtered,and the filtrate was concentrated in vacuum. The residue wascrystallized from methanol. Yield: 0.17 g, 45%.

NMR: 9.19 (s, 1H), 8.55 (d, J=2.1 Hz, 1H), 8.49-8.46 (m, 1H), 7.46-7.31(m, 4H), 7.06 (s, 1H), 6.65 (s, 1H), 5.60 (s, 2H), 2.41 (s, 3H).

MS (M+1): 383.1.

Example 7 Synthesis of Compound 78

A solution of indole (1.17 g, 10 mmol) in 10 mL tetrahydrofuran wasadded dropwise to a suspension of potassium tert-butoxide (1.34 g, 12mmol) in 10 mL tetrahydrofuran. The reaction mixture was stirred at roomtemperature for 2 hours, then 5-(chloromethyl)-1-methyl-1H-imidazole(1.31 g, 10 mmol) in 5 mL tetrahydrofuran was added dropwise. Thesolution was allowed stand for 4 hours and then 10 mL saturated ammoniumchloride was added with stirring. The mixture was extracted three timeswith a total of 60 mL of ether, the organic phase was dried usinganhydrous magnesium sulfate and filtered, and the filtrate wasconcentrated in vacuum and purified by flash chromatography on silicagel. The eluent used was a mixture of n-hexane and ethyl acetate in theratio 8:1 (vol/vol). Yield: 1.56 g, 74%.

A solution of 1-[(1-methyl-1H-5-imidazolyl)methyl]-1H-indole (211 mg,1.0 mmol) in 10 mL diethyl ether was added to oxalyl chloride (254 mg,2.0 mmol) dropwise at 0° C. The reaction mixture was stirred at 0° C.for 3 hours and then the reaction solvent was evaporated. The residuewas dissolved in 5 mL tetrahydrofuran and then the 4-bromoaniline (172mg, 1.0 mmol) and triethylamine (1 mL) in 10 mL tetrahydrofuran wasadded dropwise. The mixture was stirred for 10 hours and then 1 N NaOH(4 mL) was added to the reaction flask dropwise. The mixture wasextracted three times with a total of 60 mL of tetrahydrofuran, theorganic phase was dried using anhydrous magnesium sulfate and filtered,and the filtrate was concentrated in vacuum. The residue wascrystallized from methanol. Yield: 0.22 g, 51%.

NMR: 10.91 (br, 1H), 9.05 (s, 1H), 8.35-8.32 (m, 1H), 7.86-7.83 (m, 2H),7.70-7.7.67 (m, 2H), 7.59-7.55 (m, 3H), 7.39-7.35 (m, 2H), 6.04 (s, 2H),3.85 (s, 3H).

MS (M+1): 437.0.

Example 8 Synthesis of Compound 81

A solution of indole (1.17 g, 10 mmol) in 10 mL tetrahydrofuran wasadded dropwise to a suspension of potassium tert-butoxide (1.34 g, 12mmol) in 10 mL tetrahydrofuran. The reaction mixture was stirred at roomtemperature for 2 hours, then 4-(chloromethyl)pyridine (1.27 g, 10 mmol)in 5 mL tetrahydrofuran was added dropwise. The solution was allowedstand for 4 hours and then 10 mL saturated ammonium chloride was addedwith stirring. The mixture was extracted three times with a total of 60mL of ether, the organic phase was dried using anhydrous magnesiumsulfate and filtered, and the filtrate was concentrated in vacuum andpurified by flash chromatography on silica gel. The eluent used was amixture of n-hexane and ethyl acetate in the ratio 8:1 (vol/vol). Yield:1.35 g, 65%.

A solution of 1-(4-pyridylmethyl)-1H-indole (208 mg, 1.0 mmol) in 10 mLdiethyl ether was added to oxalyl chloride (254 mg, 2.0 mmol) dropwiseat 0° C. The reaction mixture was stirred at 0° C. for 3 hours and thenthe reaction solvent was evaporated. The residue was dissolved in 5 mLtetrahydrofuran and then the 3-methyl-5-isothiazolamine (114 mg, 1.0mmol) and triethylamine (1 mL) in 10 mL tetrahydrofuran was addeddropwise. The mixture was stirred for 10 hours and then 1 N NaOH (4 mL)was added to the reaction flask dropwise. The mixture was extractedthree times with a total of 60 mL of tetrahydrofuran, the organic phasewas dried using anhydrous magnesium sulfate and filtered, and thefiltrate was concentrated in vacuum.

The residue was crystallized from methanol. Yield: 0.17 g, 46%.

NMR: 10.34 (s, 1H), 9.18 (s, 1H), 8.60 (d, J=6.0 Hz, 2H), 8.48 (d, J=7.8Hz, 1H), 7.45-7.22 (m, 3H), 7.08 (d, J=6.0 Hz, 2H), 6.82 (s, 1H), 5.47(s, 2H), 2.47 (s, 3H).

MS (4+1): 377.

Example 9

Each of the indol-3-yl oxoacetamido compounds 1 to 102 in Table 1, wasprepared in accordance with methods described above. ¹H Nuclear magneticresonance and mass spectroscopy data for each compound was consistentwith that of the desired product.

Example 10 In Vitro Cytotoxicity Study

A panel of human cancer cells, gastric NUGC3 and HR, nasopharyngealHONE1, hepatocellular HEP G2 and HA-22T, colorectal DLD1, lung A549,prostate PC3, breast MCF7 and its adriamycin-resistant MCF7/ADR subline,uterus MES-SA and its adriamycin-resistant MES-SA/Dx5 subline, ispurchased from Food Industry Research and Development Institute,Hsinchu, Taiwan R.O.C. or American Type Culture Collection, Manassas,Va. U.S.A., are used in in vitro cytotoxicity assays. The human cellsare seeded at a cell density of 3000 or 4500 cells/100 μl/well in96-well flat-bottom plates and incubated for 24 hours at 37° C. in a 5%CO₂ incubator. The testing compounds are dissolved in dimethyl sulfoxide(DMSO) and further diluted into the culture medium for treatments ofhuman cancer cells in vitro. The drug-containing media has a final DMSOconcentration of ≦0.3%. The compounds of this invention are prepared inculture media for testing at a range of concentrations from 10, 1, 0.1,0.01 to 0.001 μM. Each compound solution is (200 μl/well in duplicate)in the cell plates and is treated for 72 hours at 37° C., 5% CO₂ in anincubator. Actinomycin D at 5 μM and 0.3% DMSO are used as the positiveand vehicle controls, respectively. A colorimetric assay using3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS) and phenazine methosulfate (PMS) is used to determine the potencyof the compounds. This assay measures cell viability based on thecellular activity in conversion of a tetrazolium salt into a coloredsoluble formazan product. The optical density (OD) values are measuredat 490 nm with a 1420 multilabel counter VICTOR® from Wallac, Turku,Finland. All of the measured values are subtracted with that of theblank control wells without cells before further calculations. Theefficacy data are expressed as a percentage normalized to the vehiclecontrols as calculated in the following formulae. Inhibitory potency (%of vehiclecontrol)=[(OD490_(compound)−OD490_(blank))/(OD490_(vehicle)−OD490_(blank))]×100%.The concentration (IC₅₀) of a test compound that inhibits 50% of thecellular activity is determined.

Results

The compounds of this invention are evaluated for cytotoxic activitiesagainst a panel of 12 human cancer cells. The concentrations (IC₅₀) of50% inhibition of 20 representative compounds of the invention are shownin Tables 1a and 1b. The compounds exhibit a broad spectrum ofanticancer activities among human cancer cell lines. Selectivity of thecompounds against different human cancer cells is observed. Likewise, avariety of compounds with diverse chemical structures also exhibitdifferential activities for a certain cancer cell type. Compound 56shows potent activities against the nasopharyngeal HONE1 cancer cell atan IC₅₀ of 3 to 4 nM, while the compound is less effective up to60-˜100-fold in other cell types.

The possibility of cross-resistance with a typical multidrug resistantcompound, adriamycin, is also studied. Certain compounds show lessefficacy in the MCF7/ADR than in MCF7. Certain compounds showessentially equal efficacy in the parent, breast MCF7 and uterus MES-SA,cancer cells as well as the adriamycin-resistant sublines implicating nocross-resistance with multi-drug resistant compounds and thus potentialclinical benefits.

TABLES 1a Anticancer activity of 20 compounds against a panel of humancancer cell lines.* Compounds DLD1 HA-22T HEP G2 HONE1 HR NUGC3 3 665510 383 174 266 279 25 543 692 1792 207 340 514 32 5650 1911 4771 9282250 4850 24 5798 2838 3594 401 5456 1715 45 593 2402 1689 48 316 465 43534 200 713 31 291 134 56 41 123 93 4 8 12 30 926 558 953 332 739 639 383720 2243 3455 487 2984 2006 65 609 302 720 50 324 242 59 658 115 613 34294 262 42 799 345 3981 296 453 634 40 981 1255 7720 56 295 395 77 1501469 >10000 380 721 458 62 651 148 6813 42 300 137 63 705 559 2473 35 78118 48 1429 303 4133 217 552 206 50 734 46 1318 38 128 37 13 846 3566310 98 396 431 54 397 382 >10000 17 64 27 *MTS assays were carried outand the data were the concentrations (IC₅₀) to inhibit 50% of the cancercell growth expressed in nM.

TABLE 1b Anticancer activity of 20 compounds against a panel of humancancer cell lines.* Com- MCF7/ MES-SA/ pounds A549 PC3 MCF7 ADR MES-SADx5 3 6397  9630  640 665 4709  6087  25 545 689 469 5241 373 398 32 NDND 5179 5291 ND** ND 24 ND ND 7651 2553 ND ND 45 611 824 611 2348 306406 43 ND ND 856 412 ND ND 56 557 628 36 5004 293 368 30 ND ND 920 847ND ND 38 ND ND 4259 4075 ND ND 65 ND ND 700 710 ND ND 59 ND ND 678 300ND ND 42 ND ND 928 527 ND ND 40 746 398 1157 1173 235 263 77 ND ND 1318595 ND ND 62 ND ND 872 509 ND ND 63 1000  636 747 5073 312 330 48 ND ND915 371 ND ND 50 ND ND 171 406 ND ND 13 ND ND 577 597 ND ND 54 1668  7255025 5410 268 333 *MTS assays were carried out and the data were theconcentrations (IC₅₀) to inhibit 50% of the cancer cell growth expressedin nM. **ND, not determined

Example 11 Evaluation of In Vivo Anticancer Activity

The in vivo anticancer activities of the compounds are evaluated by thefollowing murine leukemic P388 model. Inbred female DBA/2J mice of 4-5week-old were purchased from the Charles River Laboratories Inc.,Wilmington, Mass., USA and the National Laboratory Animals Breeding andResearch Center, Taipei, Taiwan, ROC. Murine leukemic P388 cells arepurchased from the Japanese Collection of Research Bioresources, Japan.P388 cells are cultured and propagated in RPMI1640 medium supplementedwith 50 μM 2-mercaptoethanol and 10% fetal bovine serum. Mice at the ageof 6 weeks were grouped as the treatment, negative control and positivecontrol groups at 7 to 8 mice per group. All mice are intravenouslyinoculated with the P388 cells at one million per mouse one day beforethe treatments initiated. Compounds of this invention are dissolved indimethyl sulfoxide (DMSO) and then diluted in 0.5% carboxymethylcellulose with the final concentration of DMSO less than 0.5%. Differenttreatment groups are orally given (P.O.), respectively, with compoundsof different doses for a pharmacological dose-response relationship.Mice of the negative control group are treated with the dosing vehicle,0.5% carboxymethyl cellulose. Doxorubicin of the maximal tolerated dose,10 mg/kg, is given intravenously as an experimental positive control.The cancer cell-inoculated animals are monitored twice daily. Survivalfractions of the mice are recorded and the two typical survival curvesare shown in FIGS. 1 and 2. The time on which 50% of the P388-inoculatedmice is still surviving is defined as the medium survival time and isused to calculate the percentage (normalized to the medium survival timeof the control group) of increased in life span after treatment, whichis then served as the index of treatment response.

Results and Discussion

Mice of the negative, vehicle control group consistently survive for 7to 8 days after a single inoculation of one million P388 leukemic cancercells. The experimental positive control, doxorubicin, also show aconsistent activity of prolongation of survival time in P388-inoculatedDBA/2J mice. The increased of life span by the doxorubicin treatment atits maximal tolerated dose is 119±18%, mean±sd.

Compounds 25, 56 and 48 exhibit a dose-dependent activity in theincrease of life span. The dose-response relationships of the compoundsagainst the leukemic cancer are summarized in Table 2. It is also notedthat the continuation of daily oral treatment with these compoundsshowed a significant longer survival of the mice. No significanttoxicity evident in the body weight loss is observed for the repeatedlydosing regimens through the oral route. The P388 leukemic cancer modelresults demonstrate the in vivo anticancer activity of the compoundsafter oral administration.

TABLE 2 compound 25 56 48  25 mg/kg, P.O.*, daily, 4× 14 ± 1% 26 ± 27%25%  50 mg/kg, P.O., daily, 4× 27 ± 3% 57 ± 20% 60% 100 mg/kg, P.O.,daily, 4× 20 ± 8% 63 ± 17% 25% 150 mg/kg, P.O., daily, 4× ND** ND 68 ±25% 200 mg/kg, P.O., daily, 4×  46 ± 24% 100 ± 21%  63% 400 mg/kg, P.O.,daily, 4× ND ND 229%  *P.O., oral administration **ND, not determined

Example 12 Evaluation of Angiogenesis Inhibition

Establishment of Rat Aorta Tube Formation Assay

The procedures are modified from previous reports (Burbridge and West2001; Bauer et al. 2000; Nicosai and Ottinetti 1990a) and described asfollows. Male Spraque-Dawley rats of 6-8 weeks old are purchased fromthe National Laboratory Animal Breeding and Research Center (Taipei,Taiwan). The animals are anesthetized with urethane (2 g/kg,intraperitoneal) and the thoracic aortas are quickly harvested in alaminar flow hood. The aortas are rinsed with ice-cold normal salinewith 10 μg/ml gentamicin and the attached fibroadipose tissues areremoved using micro-dissection scissors. The aortas are cross-sectionedinto rings of 1 mm in thickness. A mixture of Matrigel®, purchased fromBD Biosciences, solution and the EBM-II medium, purchased fromBioWhittaker Inc., (1:1) with 2% FBS, 0.25 mg/ml, amphotericin B and 10μg/ml gentamicin is prepared and added at 100 μl to each well of a48-well plate. The mixture is allowed to gel at 37° C. Aorta rings arethen placed onto the gelled matrix and embedded in an additional 150 μlper well of the mixture solution and allowed to gel at 37° C. The aortarings are then covered with 125 μl of the EGM-II medium and incubatedovernight in humidified incubator with 5% CO₂/95% air at 37° C. Afterremoval of the EGM-II from the established aorta cultures, thecompound-containing media are applied at 300 μl per well to the culturesand then incubated in 5% CO₂/95% air at 37° C. for 5 days. The culturesare then subject to the 3-[4,5-dimethylthiazol-2-yl]-2,5diphenyl-2H-tetrazolium bromide (MTT) or3-(4,5-dimethylthiazol-2-yl)-5-(3-carbomethoxyphenyl)-2H-tetrazolium(MTS) assay and OD490 nm values measured. The testing compounds aredissolved in dimethyl sulfoxide (DMSO) and prepared in EBM-II media withat a final concentration of DMSO less than 0.1%.

MTT Staining

The capillary-like, tube structure of the outgrown rat aorta endothelialcells is easily visualized by MTT staining. The medium on top of thematrix is removed from the established aorta ring cultures. MTT solutionof 5 mg/ml in saline is added to the cultures at 100 μl/well and thecultures are incubated in a CO₂ incubator at 37° C. for 24 hr. Thestained cultures are then examined under a microscope DMIRB from Leicaand photos taken with a digital camera.

MTS Assay

Inhibition activity is also assessed in the following manner. Thereductive reaction of MTS initiated by the viable cellular mitochondrialactivity is used as a basis for the measurement of cell viability (Coryet al. 1991; Gieni et al. 1995). To the rat aorta ring cultures 300 μlof a solution containing 100 μg MTS and 1 μg, phenazine methosulfate isadded per well and incubated in 5% CO₂/95% air at 37° C. for 24 hr.During the incubation, MTS is converted to formazan product. At the endof incubation, the solution overlaying the gel is well-mixed and 100 μlaliquots are transferred into a 96-well plate for measurement ofOD_(490 nm) with a spectrophotometer VICTOR™-II from Wallac Oy.

The compounds listed in Table 3 are screened for angiogenesisinhibition. The control compound, ASTA, is D-24851,N-[pyridin-4-yl]-[1-(4-chlorobenzyl)-indol-3-yl]-glyoxylic acid amide.Noninhibitors are indicated by the symbol (−), meaning that the extentof endothelial cell growth is essentially at the same level as that ofthe control. The most potent inhibitors receive a score of (+++++),indicating essentially no measurable endothelial cell growth.

TABLE 3 Final Inhibition Compound conc. Score ASTA compound   1 μg/ml++++  3 0.3 μg/ml +++++ 25 0.3 μg/ml +++++ 32 0.3 μg/ml +++++ 24 0.3μg/ml − 45 0.3 μg/ml +++++ 56 0.3 μg/ml +++++ 38 0.3 μg/ml − 27   1μg/ml +++ 65 0.3 μg/ml +++++ 12   1 μg/ml +++ 56   1 μg/ml − 59 0.3μg/ml +++++ 42 0.3 μg/ml +++++ 40 0.3 μg/ml +++++ 77 0.3 μg/ml +++++ 620.3 μg/ml ++ 63 0.3 μg/ml +++++ 13 0.3 μg/ml +++++ 54 0.3 μg/ml +++++ 44  1 μg/ml +++++

Example 13 Evaluation of Antitumor Activity

Human colorectal SW480 cancer cells are subcutaneously implanted intothe young male athymic nude mice at 1 million cells per mouse. Thetreatment schedule of 200 mg/kg daily oral administration for 6 weeks isinitiated at the time which the tumor size reaches 50˜100 mm³. Compound56 is orally gavaged to the nude mice subcutaneously bearing humancolorectal SW480 tumor xenograft based on the treatment schedule. Thetumor size and body weight of the animals are monitored twice a weekthrough out the 70-day period of observation. The tumor growth issuppressed by the daily oral treatments of compound 56. Tumor sizes ofthe treated and vehicle control groups are compared at the end of 70-dayobservation. The size of tumor mass subcutaneously implanted in the nudemice for the treated group is reduced to 26% compared to that of thevehicle control group at the end of the observation period. There is noobvious toxicity as noted by no significant loss of the body weight forthe oral multiple dosing treatment.

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A method of treating cancer, the cancer being leukemia or colorectalcancer, comprising administering to a subject in need thereof aneffective amount of a compound of the formula 1:

wherein, R¹ is isoxazolyl, thiazolyl, isothiazolyl, 1,3,4-thiadiazolyl,or 1,3-benzothiazolyl, each being optionally substituted with 1-6independent R⁵; R² is H; R³ is C1-C10 alkyl, C2-C10 alkenyl, C2-C10alkynyl, C3-C10 cycloalkyl, C4-C10 cycloalkenyl, isoxazolyl, furanyl,thiophenyl, thiazolyl, imidazolyl, or pyridyl, each being optionallysubstituted with 1-4 independent R⁵; each R⁴ is independently H, NO₂,halo, CN, R⁷, or OR⁷; each n is 0, 1, 2, 3, or 4; each R⁵ isindependently H, C1-C10 alkyl optionally substituted with 1-4independent R⁶, C2-C10 alkenyl, C2-C10 alkynyl, C3-C10 cycloalkyl, aryloptionally substituted with 1-4 independent R⁶, heteroaryl optionallysubstituted with 1-4 independent R⁶, halo, or haloalkyl; each R⁶ isindependently C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, halo,haloalkyl, CN, NO₂, OR⁷, or SO₂R⁷; and each R⁷ is independently H,C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, C3-C10 cycloalkyl, aryl,or heteroaryl.
 2. The method of claim 1, wherein R¹ is isothiazolyl,isoxazolyl, or thiazolyl, each being optionally substituted with 1-4C1-C10 alkyl.
 3. The method of claim 1, wherein R¹ is3-methyl-5-isothiazolyl.
 4. The method of claim 1, wherein R³ is3-methyl-5-isoxazolyl, 3-phenyl-5-isoxazoyl, pyridyl, or thiophenyl. 5.The method of claim 1, wherein R¹ is 3-methyl-5-isothiazolyl,3-methyl-5-isoxazolyl, 3-phenyl-5-isoxazolyl, 3-tert-butyl-5-isoxazolyl,4-methyl-1,3-thiazol-2-yl, or 1,4-benzodioxan-6-yl, thiazolyl; and R³ is3-methyl-5-isoxazolyl or thiophenyl.
 6. The method of claim 1, whereinthe compound is


7. The method of claim 1, wherein the cancer is leukemia.
 8. The methodof claim 1, wherein the cancer is colorectal cancer.